Adamts binding immunoglobulins

ABSTRACT

The present invention relates to immunoglobulins that bind ADAMTSS and more in particular to polypeptides, that comprise or essentially consist of one or more such immunoglobulins. The invention also relates to constructs comprising such immunoglobulins, such as immunoglobulin single variable domains (ISVDs) or polypeptides as well as nucleic acids encoding such immunoglobulins or polypeptides; to methods for preparing such immunoglobulins, polypeptides and constructs; to host cells expressing or capable of expressing such immunoglobulins or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such immunoglobulins, polypeptides, constructs, nucleic acids and/or host cells; and to uses of immunoglobulins, polypeptides, constructs, nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes mentioned herein. Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

FIELD OF THE INVENTION

The present invention relates to immunoglobulins that bind ADAMTS5 andmore in particular to polypeptides, that comprise or essentially consistof one or more such immunoglobulins (also referred to herein as“immunoglobulin(s) of the invention”, and “polypeptides of theinvention”, respectively). The invention also relates to constructscomprising such immunoglobulins, such as immunoglobulin single variabledomains (ISVDs) or polypeptides as well as nucleic acids encoding suchimmunoglobulins or polypeptides (also referred to herein as “nucleicacid(s) of the invention”; to methods for preparing suchimmunoglobulins, polypeptides and constructs; to host cells expressingor capable of expressing such immunoglobulins or polypeptides; tocompositions, and in particular to pharmaceutical compositions, thatcomprise such immunoglobulins, polypeptides, constructs, nucleic acidsand/or host cells; and to uses of immunoglobulins, polypeptides,constructs, nucleic acids, host cells and/or compositions, in particularfor prophylactic and/or therapeutic purposes, such as the prophylacticand/or therapeutic purposes mentioned herein. Other aspects,embodiments, advantages and applications of the invention will becomeclear from the further description herein.

BACKGROUND OF THE INVENTION

Osteoarthritis (OA) is one of the most common causes of disabilityworldwide. It affects 30 million Americans and is the most common jointdisorder. It is projected to affect more than 20 percent of the U.S.population by 2025. The disease is non-systemic and is usuallyrestricted to few joints. However, the disease can occur in all joints,most often the knees, hips, hands, shoulder and spine. OA ischaracterized by progressive erosion of articular cartilage (cartilagethat covers the bones) resulting in chronic pain and disability.Eventually, the disease leads to total destruction of the articularcartilage, sclerosis of underlying bone, osteophyte formation etc., allleading to loss of movement and pain. Osteoarthritis can be defined as adiverse group of conditions characterised by a combination of jointsymptoms, signs stemming from defects in the articular cartilage andchanges in adjacent tissues including bone, tendons and muscle. Pain isthe most prominent symptom of OA and most often the reason patients seekmedical help. There is no cure for OA, i.e. current treatments do notinhibit structural deterioration of the OA joint. Disease management islimited to treatments that are palliative at best and do little toaddress the underlying cause of disease progression. Although diseaseinitiation may be multi-factorial, the cartilage destruction appears tobe a result of uncontrolled proteolytic destruction of the extracellularmatrix (ECM). The most abundant ECM components of articular cartilageare Collagen (foremost Collagen II) and the proteoglycans, mainlyAggrecan (Kiani et al. 2002 Cell Research 12:19-32).

Aggrecan is important in the proper functioning of the articularcartilage because it provides a hydrated gel structure that endows thecartilage with load-bearing properties. Aggrecan is a large,multimodular molecule (2317 amino acids) expressed by chondrocytes. Itscore protein is composed of three globular domains (G1, G2 and G3) and alarge extended region between G2 and G3 for glycosaminoglycan chainattachment. This extended region comprises two domains, one substitutedwith keratan sulfate chains (KS domain) and one with chondroitin sulfatechains (CS domain). The CS domain has 100-150 glycosaminoglycan (GAG)chains attached to it. Aggrecan forms large complexes with Hyaluronan inwhich 50-100 Aggrecan molecules interact via the G1 domain and LinkProtein with one Hyaluronan molecule. Upon uptake of water (due to theGAG content) these complexes form a reversibly deformable gel thatresists compression. The structure, fluid retention and function ofjoint cartilage is linked to the matrix content of Aggrecan, and theamount of chondroitin sulfate bound to the intact core protein.Structurally, OA is characterized by degradation of Aggrecan,progressively releasing domains G3 and G2 (resulting in ‘deflation’ ofthe cartilage) and eventually release of the G1 domain and degradationof Collagen, irreversibly destroying the cartilage structure. The mostsignificant Aggrecan cleavage site for OA pathogenesis is located at thesequence TEGE^(373↓374)ARGS. This cleavage site is positioned in theinterglobular domain (IGD) of Aggrecan. Antibodies that recognize the³⁷⁴ARGS neo-epitope led to the discovery of aggrecanase-1, which provedto be ADAMTS4 and aggrecanase-2, which is ADAMTS5. Subsequently, otherrelated ADAMTS enzymes, including ADAMTS1, -8, -9, -15 and -20, wereshown to have aggrecanase activity. ADAMTS16 and 18 are also weakaggrecanases. The ADAMTS5 and matrix metalloproteinases (MMPs) share abinding site to Aggrecan that is very similar both in sequence and inoverall shape (El Bakali et a. 2014 Future Medicinal Chemistry (Review)6:1399).

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondinmotifs) enzymes are secreted, multi-domain matrix-associated zincmetalloendopeptidases that have diverse roles in tissue morphogenesisand patho-physiological remodeling, in inflammation and in vascularbiology (Kelwick et al. 2015 Genome Biology 16:113). The human familyincludes 19 members that can be sub-grouped on the basis of their knownsubstrates. The aggrecanases or proteoglycanases include ADAMTS1, -4,-5, -8, -9, -15 and -20, which can cleave hyaluronan-binding chondroitinsulfate proteoglycan (CSPG) extracellular proteins, including Aggrecan,versican, brevican and neurocan. The two most preferred cleavage sitesin bovine Aggrecan are at KEEE^(1657↓1668)GLGS, followed byGELE^(1480↓1481)GRGT. Thereafter, cleavage occurs atNITEGE^(373↓374)ARGS in the IGD (at which MMPs do not cut), releasingthe afore-mentioned neo-epitope, and at TAQE^(1771↓1772)AGEG andVSQE^(1871↓1872)LGQR in the CS-2 region (Fosang et al. 2008 EuropeanCells and Materials 15:11-26). These cleavage sites are highly conservedin humans, bovine, mice and rats.

Various lines of evidence indicate that ADAMTS5 is a principal enzymeinvolved in the pathogenesis of osteoarthritis. ADAMTS5 is a majoraggrecanase present in cartilage. In human cartilage explants andchondrocytes, knockdown of ADAMTS5 attenuated Aggrecan breakdown,suggesting that this enzyme may be involved in human tissues. Expressionof the enzyme is augmented by cytokines such as interleukin-1 andoncostatin-M, which provoke Aggrecan breakdown in tissues. ADAMTS5generated Aggrecan fragments are detected in the synovial fluid andserum of OA patients (Germaschewski et al., 2014 OsteoarthritisCartilage 22:690-697).

ADAMTS5 is first synthesized as an inactive protein, including aprotease domain at the N-terminus and an ancillary domain at theC-terminus. The protease domain consists of a signal peptide, aprodomain with a furin recognition sequence and a catalytic domain. Theprodomain is cleaved by proprotein convertases in order to produce theactive enzymes. ADAMTS5 further contains ancillary domains whichactively participate in substrate recognition and modulate the affinityof the proteinase for its substrate(s) (“exosites”). Thedisintegrin-like domain, central thrombospondin type I-like (TS) repeat,cysteine-rich domain, spacer region and additional TS motif of ADAMTS5are ancillary domains with potential exosite functions. Thecysteine-rich domain appears to be essential for the binding and dockingof ADAMTS5 onto glycosaminoglycans. The greatest variability in theADAMTS members is found in these ancillary domains (Kelwick et al., 2015Genome Biology 16:113).

Disease modifying anti-osteoarthritic drugs (DMOADs), which can bedefined as drugs that inhibit structural disease progression and ideallyalso improves symptoms and/or function are intensely sought after.DMOADs are likely to be prescribed for long periods in this chronicillness of an aging population, therefore demanding excellent safetydata in a target population with multiple comorbidities and thepotential for drug-drug interactions.

Several pharmaceutical companies have developed small moleculeinhibitors of ADAMTS5. Some of these compounds are claimed to bespecific for ADAMTS5, whereas others have effect also against otherADAMTS members, or even against MMPs. These cross-inhibitions areconsidered to be responsible for musculoskeletal syndrome, a side effectcaused by broad-spectrum inhibitors and involving arthralgia, myalgia,joint stiffness and tendonitis (Santamaria et al., 2015 Biochem J471:391-401). The—Wyeth aggrecanase inhibitor AGG-523 was used in 5phase I clinical trials in healthy subjects and patient with OA, but hasnot been taken further. Nor have the other small molecule ADAMTSinhibitors entered any further clinical development as potential DMOAD(Bondeson et al., 2015 Drug Discovery 10:5-14). Indeed, despite a numberof recent clinical trials specifically investigating DMOADs, no suchtreatments have been approved so far (El Bakali et al., 2014 FutureMedicinal Chemistry (Review) 6:1399).

In view of the success of targeted biologic therapy using antibodies(“Abs”), there was interest in developing similar therapeutic strategiesfor OA. A study of the Rottapharm monoclonal antibody (mAb) CRB0017,directed against the spacer domain of ADAMTS5, showed that in mice,intra-articular administration of this mAb significantly preventeddisease progression in a dose-dependent manner (Chiusaroli et al., 2013Osteoarthritis Cartilage 21:1807). There was no comparison with systemicadministration, nor was it assessed to what degree the mAb leaked fromthe synovial space. Another study used systemic administration of themAb 12F4 in mice, which demonstrated both structural diseasemodification and alleviation of pain-related behaviour (Miller et al.,2014 Osteoarthritis Cartilage 22iii, S35). However, a singleadministration of mAb 12F4 in cynomolgus monkey caused focalhaemorrhage, a dose-dependent increased mean arterial pressure andcardiac conductance abnormalities (more specifically, ST elevations andventricular arrhythmias on the ECG) indicating cardiac ischemia, whichwere sustained for up to 8 months after administration of the singledose (Larkin et al., 2014 Osteoarthritis Cartilage 22iii, S483). Theseside effects halted further clinical development of mAb 12F4.

WO2008/074840 in the name of Ablynx NV describes the generation ofNanobodies® against members of the A Disintegrin and Metalloproteinases(ADAM) family, including ADAMTS5.

Therapeutic interventions in joints have further been hindered by thedifficulty of targeting drugs to articular cartilage. Because articularcartilage is an avascular and alymphatic tissue, traditional routes ofdrug delivery (oral, intravenous, intramuscular) ultimately rely ontranssynovial transfer of drugs from the synovial capillaries tocartilage by passive diffusion. This prompted the development ofintra-articular (IA) delivery of medicaments.

On the other hand, IA delivery of therapeutic proteins has been limitedby their rapid clearance from the joint space and lack of retentionwithin cartilage; and restriction to large joints. Synovial residencetime of a drug in the joint is often less than 24 h (Edwards 2011 Vet J190:15-21; Larsen et al., 2008 J Pham Sci 97:4622-4654). Due to therapid clearance of most IA injected drugs, frequent injections would beneeded to maintain an effective concentration (Owen et al., 1994 Br JClin Pharmacol 38:349-355). Moreover, IA delivery of therapeuticproteins is not feasible practically for small joints, which hamperstreatment of e.g. OA-fingers.

There remains a need for effective DMOADs.

SUMMARY OF THE INVENTION

The present invention aims to provide polypeptides against OA withimproved prophylactic, therapeutic and/or pharmacological properties, inaddition to other advantageous properties (such as, for example,improved ease of preparation, good stability, and/or reduced costs ofgoods), compared to the prior art amino acid sequences and antibodies.In particular, the present invention aims to provide polypeptidesinhibiting ADAMTS and especially inhibiting ADAMTS5.

The ADAMs, ADAMTSs and MMPs share a binding site to Aggrecan that isvery similar both in sequence and in overall shape. Since the variousother ADAM (including TACE) and ADAMTS family members have diverse rolesin normal physiology are strongly associated with many commonpathological conditions, including asthma, arthritis, cancer, connectivetissue disorders or thrombotic thrombocytopenic purpura (El Bakali etal., supra), the inventors appreciated the importance of preservingselectivity. In order to effectively silence only ADAMTS5 activity,targeting the catalytic domain appeared to be the best option. However,the catalytic domains of ADAMTS4 and ADAMTS5 share a high degree ofsequence similarity (cf. El Bakali et al, supr). In addition, it turnedout that the high sequence conservation of the catalytic domain betweenvarious species foregoes a robust immune response.

Surprisingly, the ISVDs of the invention met these two seeminglymutually exclusive requirements of inhibiting the (enzymatic) activityof ADAMTS5 on the one hand, while preserving selectivity on the otherhand.

Various monovalent ISVDs of the present invention were equipotent to theconventional bivalent antibody mAb 12F4 H4L0 in an AlphaLISA enzymaticassay. In addition thereto, the ISVDs of the present invention were alsoequipotent to the conventional bivalent antibody mAb12F4 H4L0 at high oreven excess Aggrecan substrate concentration, which is reminiscent ofthe joints. On the other hand, in an ex vivo bovine explant assay, whichresembles even more closely physiological conditions, most monovalentISVDs had a better IC₅₀ than the comparator mAb 12F4 H4L0 (“12F4”) andmAb CRB0017 of Rottapharm. In a human ex vivo explant assay, the ISVDsof the present invention also were substantially better than the priorart conventional antibody CRB0017, as demonstrated by IC₅₀.

After further engineering the ISVDs in view of various diverse andfavorable features, including stability, affinity and inhibitoryactivity as well as minimizing immunogenicity, these ISVDs were nextevaluated in vivo.

Systemic administration of the ISVDs of the present inventiondemonstrated potent inhibition of the aggrecanase activity in vivo asevaluated in cynomolgus monkey. In addition, the ISVDs were also safe touse, in contrast to the prior art antibody 12F4. Also in an in vivomouse medial meniscal destabilization (DMM) model, the ISVDs of thepresent invention showed a structural benefit up to 50% for bothprophylactic as well as therapeutic treatment by systemicadministration. Early treatment with the ISVDs of the present inventioncaused a dose-dependent, significant and meaningful symptomatic benefitduring Anterior Cruciate Ligament Transection and Resection of themedial meniscus (ACLT+tMx) induced OA in rats.

The ISVDs are eventually intended for inhibiting ADAMTS5 in the jointsand therefore will need to resist the conditions of synovial fluid inthe joints, which contains various proteases. Next to the favourablecharacteristics of above, it was shown that the isolated ISVDs wereextremely stable in synovial fluid. It is anticipated that thisstability enables a less frequent dosage regimen.

Accordingly, the present invention relates to a polypeptide comprisingat least 1 immunoglobulin single variable domain (ISVD) binding an ADisintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS),preferably wherein said ADAMTS is chosen from the group consisting ofADAMTS1-ADAMTS9, preferably ADAMTS5, ADAMTS4, ADAMTS1, ADAMTS8, ADAMTS9and ADAMTS15 and ADAMTS20, most preferably ADAMTS5. In an aspect, theinvention relates to a polypeptide as described herein, wherein saidISVD binding ADAMTS, preferably ADAMTS5 does not bind ADAMTS4, MMP1 orMMP14.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD specifically binding ADAMTS5essentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 21,35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; and aminoacid sequences that have 1, 2 or 3 amino acid difference(s) with SEQ IDNOs: 21, 35, 20, 22, 25, 33, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and34; (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 37, 53,36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52;and amino acid sequences that have 1, 2 or 3 amino acid difference(s)with SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119,42, 46, 47, 48, 49 and 52; and (iii) CDR3 is chosen from the groupconsisting of SEQ ID NO: SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62,63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70; and amino acid sequencesthat have 1, 2, 3 or 4 amino acid difference(s) with SEQ ID NOs: 55,118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and70.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD specifically binding ADAMTS5essentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which (i) CDR1 is chosen from the group consisting of (a) SEQ ID NO:22; and (b) amino acid sequence that has 1, 2, 3, 4, 5 or 6 amino aciddifference(s) with SEQ ID NO: 22, wherein at position 2 the S has beenchanged into R; at position 3 the A has been changed into T; at position4 the V has been changed into F; at position 6 the V has been changedinto S; at position 7 the N has been changed into Y; and/or at position10 the A has been changed into G; (ii) CDR2 is SEQ ID NO: 36; and (iii)CDR3 is SEQ ID NO: 54.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD specifically binding ADAMTS5essentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which (i) CDR1 is SEQ ID NO: 33; (ii) CDR2 is chosen from the groupconsisting of (c) SEQ ID NO: 50; and (d) amino acid sequence that has 1,2, or 3 amino acid difference(s) with SEQ ID NO: 50, wherein at position8 the M has been changed into I; at position 9 the P has been changedinto T; and/or at position 10 the Y has been changed into F; and (iii)CDR3 is chosen from the group consisting of (e) SEQ ID NO: 68; and (f)amino acid sequence that has 1 or 2 amino acid difference(s) with SEQ IDNO: 68, wherein at position 5 the F has been changed into L; and/or atposition 11 the D has been changed into E.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD specifically binding ADAMTS5essentially consists of 4 framework regions (FR1 to FR4, respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which (i) CDR1 is SEQ ID NO: 28; (ii) CDR2 is chosen from the groupconsisting of (c) SEQ ID NO: 44; and (d) amino acid sequence that has 1,2, or 3 amino acid difference(s) with SEQ ID NO: 44, wherein at position3 the S has been changed into T; at position 4 the R has been changedinto W; at position 8 the T has been changed into I; and/or at position9 the T has been changed into L; and (iii) CDR3 is chosen from the groupconsisting of (e) SEQ ID NO: 62; and (f) amino acid sequence that has 1or 2 amino acid difference(s) with SEQ ID NO: 62, wherein at position 1the G has been changed into S; and/or at position 14 the D has beenchanged into E.

In preferred embodiments of all aspects of the invention animmunoglobulin single variable domain (ISVD) according to the inventionpreferably consists of or essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regionsCDR1, CDR2 and CDR3 as outlined herein above and below. Preferredframework sequences are disclosed for example in the table A-2 below andcan be used in an ISVD of the invention. Preferably, the CDRs depictedin Table A-2 are matched with the respective framework regions of thesame ISVD construct.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD is chosen from the group of ISVDs,wherein: CDR1 is chosen from the group consisting of SEQ ID NOs: 21, 35,20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; CDR2 ischosen from the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51,44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and CDR3 ischosen from the group consisting of SEQ ID NOs: 55, 118, 71, 54, 58, 68,69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and 70.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ISVD is chosen from the group of ISVDs,wherein:

-   -   CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37 and CDR3 is SEQ ID        NO: 55;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53 and CDR3 is SEQ ID        NO: 118;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53 and CDR3 is SEQ ID        NO: 71;    -   CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36 and CDR3 is SEQ ID        NO: 54;    -   CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36 and CDR3 is SEQ ID        NO: 54;    -   CDR1 is SEQ ID NO:25, CDR2 is SEQ ID NO:40 and CDR3 is SEQ ID        NO:58;    -   CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 50 and CDR3 is SEQ ID        NO: 68;    -   CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51 and CDR3 is SEQ ID        NO: 69;    -   CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 44 and CDR3 is SEQ ID        NO: 62;    -   CDR1 is SEQ ID NO:28, CDR2 is SEQ ID NO:45 and CDR3 is SEQ ID        NO:63;    -   CDR1 is SEQ ID NO:28, CDR2 is SEQ ID NO:43 and CDR3 is SEQ ID        NO:61;    -   CDR1 is SEQ ID NO:24, CDR2 is SEQ ID NO:39 and CDR3 is SEQ ID        NO:57;    -   CDR1 is SEQ ID NO:23, CDR2 is SEQ ID NO:38 and CDR3 is SEQ ID        NO:56;    -   CDR1 is SEQ ID NO:26, CDR2 is SEQ ID NO:41 and CDR3 is SEQ ID        NO:59;    -   CDR1 is SEQ ID NO:27, CDR2 is SEQ ID NO:119 and CDR3 is SEQ ID        NO:60;    -   CDR1 is SEQ ID NO:27, CDR2 is SEQ ID NO:42 and CDR3 is SEQ ID        NO:60;    -   CDR1 is SEQ ID NO: 29, CDR2 is SEQ ID NO: 46 and CDR3 is SEQ ID        NO: 64;    -   CDR1 is SEQ ID NO:30, CDR2 is SEQ ID NO:47 and CDR3 is SEQ ID        NO:65;    -   CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48 and CDR3 is SEQ ID        NO: 66;    -   CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49 and CDR3 is SEQ ID        NO: 67; and    -   CDR1 is SEQ ID NO: 34, CDR2 is SEQ ID NO: 52 and CDR3 is SEQ ID        NO: 70.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, in which CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37and CDR3 is SEQ ID NO: 55.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, in which said ISVD is chosen from the group consistingof SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117,12, 13, 14, 15 and 18.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide binds to ADAMTS5 with a K_(D)between 1 E⁻⁰⁷M and 1 E⁻¹³ M, such as between 1 E⁻⁰⁸ M and 1 E⁻¹² M,preferably at most 1 E⁻⁰⁷ M, preferably lower than 1 E⁻⁰⁸ M or 1 E⁻⁰⁹ M,or even lower than 1 E⁻¹⁰ M, such as 5 E⁻¹¹ M, 4 E⁻¹¹ M, 3 E⁻¹¹ M, 2E⁻¹¹ M, 1.7 E⁻¹¹ M, 1 E⁻¹² M, or even 5 E⁻¹² M, 4 E⁻¹² M, 3 E⁻¹²M, 1E⁻¹² M, for instance as determined by KinExA, or alternatively byGyrolab.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide inhibits an activity ofADAMTS5 with an IC₅₀ between 1 E⁻⁰⁷ M and 1 E⁻¹³ M, such as between 1E⁻⁰⁸ M and 1 E⁻¹¹ M, for instance as determined by human FRET assay orhuman AlphaLSA.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide inhibits an (enzymatic)activity of ADAMTS5 with an IC₅₀ of at most 1 E⁻⁰⁷ M, preferably 1 E⁻⁰⁸M, 5 E⁻⁰⁹ M, or 4 E⁻⁹ M, 3 E⁻¹¹ M, 2 E⁻⁹ M, such as 1 E⁻⁹ M.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide modulates ADAMTS5 with anEC₅₀ between 1 E⁻⁰⁷ M and 1 E⁻¹² M, such as between 1 E⁻⁰⁸M and 1 E⁻¹¹M, for instance as determined by binding ELISA.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide binds to ADAMTS5 with anoff-rate of less than 1 E⁻⁰⁴ s⁻¹, for instance as determined by SPR.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said ADAMTS5 is human ADAMTS5 (SEQ ID NO:149), bovine ADAMTS5 (SEQ ID NO: 150), rat ADAMTS5 (SEQ ID NO: 151),guinea pig ADAMTS5 (SEQ ID NO: 152), mouse ADAMTS5, (SEQ ID NO: 153) orcynomolgus ADAMTS, (SEQ ID NO: 154), preferably human ADAMTS5, mostpreferably SEQ ID NO: 149.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide antagonizes an activity ofADAMTS5, such as a protease activity, such as cleavage of Aggrecan,versican, brevican, neurocan, decorin, and/or biglycan, preferablycleavage of Aggrecan; preferably antagonizes aggrecanase activity ofADAMTS5.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide blocks the binding of ADAMTS5to Aggrecan of at least 20%, such as at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or even more, for instance as determined by FRET,AlphaLISA or ELISA.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide inhibits the proteaseactivity of ADAMTS5, such as inhibits the proteolysis of a substrate,such as Aggrecan, versican, brevican, neurocan, decorin, and/orbiglycan, preferably Aggrecan.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, comprising at least 2 ISVDs, wherein at least 1 ISVDspecifically binds ADAMTS, preferably ADAMTS5, preferably chosen fromthe group consisting of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11,9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18, preferably wherein said atleast 2 ISVDs specifically bind ADAMTS, preferably ADAMTS5.

In a further preferred aspect, the invention relates to a polypeptidecomprising two or more ISVDs which specifically bind ADAMTS5, wherein(a) at least a “first” ISVD specifically binds a first antigenicdeterminant, epitope, part, domain, subunit or conformation of ADAMTS5;and wherein (b) at least a “second” ISVD specifically binds a secondantigenic determinant, epitope, part, domain, subunit or conformation ofADAMTS5, different from the first antigenic determinant epitope, part,domain, subunit or conformation, respectively, preferably wherein said“first” ISVD specifically binding ADAMTS5 is chosen from the groupconsisting of SEQ ID NO:s 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8,117, 12, 13, 14, 15 and 18, preferably wherein said “second” ISVDspecifically binding ADAMTS5 is SEQ ID NO: 118 or 19, even morepreferably said polypeptide is chosen from the group consisting of SEQID NO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 1292F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128(clone 1319D3-093-Alb).

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, further comprising an ISVD binding serum albumin,preferably wherein said ISVD binding serum albumin essentially consistsof 4 framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which CDR1 is SEQ IDNO: 146, CDR2 is SEQ ID NO: 147, and CDR3 is SEQ ID NO: 148, even morepreferably wherein said ISVD binding serum albumin is chosen from thegroup consisting of ALB8 (SEQ ID NO: 131), ALB23 (SEQ ID NO: 132),ALB129 (SEQ ID NO: 133), ALB132 (SEQ ID NO: 134), ALB11 (SEQ ID NO:135), ALB11 (S112K)-A (SEQ ID NO: 136), ALB82 (SEQ ID NO: 137), ALB82-A(SEQ ID NO: 138), ALB82-AA (SEQ ID NO: 139), ALB82-AAA (SEQ ID NO: 140),ALB82-G (SEQ ID NO: 141), ALB82-GG (SEQ ID NO: 142), ALB82-GGG (SEQ IDNO: 143), ALB92 (SEQ ID NO: 144), and ALB223 (SEQ ID NO: 145), even morepreferably wherein said polypeptide is chosen from the group consistingof SEQ ID NO: 129 (clone 577 2F3^(SO)-Alb), SEQ ID NO: 130 (clone 5792F3^(SO)-093-Alb), SEQ ID NO: 120 (clone 4 2A12-Alb), SEQ ID NO: 121(clone 5 2D7-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), SEQ ID NO: 123(clone 69 049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125(clone 713B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO:127 (clone 130 049-093-Alb), and SEQ ID NO: 128 (clone 1319D3-093-Alb).

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, further comprising at least one ISVD specificallybinding Aggrecan, preferably chosen from the group consisting of SEQ IDNO: 156 (Nanobody 00745 PEA114F08) and SEQ ID NO: 157 (Nanobody 00747PEA604F02).

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, comprising at least 2 ISVDs specifically bindingAggrecan, wherein said at least 2 ISVDs specifically binding Aggrecancan be the same or different, preferably wherein said at least 2 ISVDsspecifically binding Aggrecan are independently chosen from the groupconsisting of SEQ ID NOs: 156 and 157, even more preferably wherein saidISVD specifically binding Aggrecan, specifically binds to human Aggrecan[SEQ ID NO: 155]. Preferably, said ISVD specifically binding Aggrecan,specifically binds dog Aggrecan (see also table 2), bovine Aggrecan, ratAggrecan; pig Aggrecan; mouse Aggrecan, rabbit Aggrecan; cynomolgusAggrecan and/or rhesus Aggrecan. Preferably, said ISVD specificallybinding Aggrecan preferably binds to cartilaginous tissue such ascartilage and/or meniscus.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide has a stability of at least 7days, such as 14 days, 21 days, 1 month, 2 months or even 3 months insynovial fluid (SF) at 37° C.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said at least two ISVDs are directly linked toeach other or are linked via a linker, preferably said linker is chosenfrom the group consisting of SEQ ID NOs: 158 to 174 (i.e. SEQ ID NO:158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173 and 174), preferably SEQ ID NO: 169.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, further comprising a C-terminal extension, preferablywherein said C-terminal extension is a C-terminal extension (X)n, inwhich n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (andpreferably 1 or 2, such as 1); and each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen, andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I).

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide has at least 80%, 90%, 95% or100% sequence identity with any of SEQ ID NOs: 1-19 (i.e. SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19),116-117 or 120-130 (i.e. SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126,127, 128, 129 and 130).

In a further preferred aspect, the invention relates to a method oftreating and/or preventing diseases or disorders in an individual, forinstance in which ADAMTS5 activity is involved, the method comprisingadministering the polypeptide according to any one of claims 1 to 43 tosaid individual in an amount effective to treat or prevent a symptom ofsaid disease or disorder, preferably wherein said diseases or disordersis chosen from the group consisting of arthropathies andchondrodystrophies, arthritic disease, such as osteoarthritis,rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumaticrupture or detachment, achondroplasia, costochondritis,Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar diskdegeneration disease, degenerative joint disease, and relapsingpolychondritis, osteochondritis dissecans and aggrecanopathies. Morepreferably, said disease or disorder is an arthritic disease and mostpreferably osteoarthritis.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, for use as a medicament.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, for use in treating or preventing a symptom of anADAMTS5 associated disease, such as e.g. arthropathies andchondrodystrophies, arthritic disease, such as osteoarthritis,rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumaticrupture or detachment, achondroplasia, costochondritis,Spondylo-epimetaphyseal dysplasia, spinal disc herniation, lumbar diskdegeneration disease, degenerative joint disease, and relapsingpolychondritis, osteochondritis dissecans and aggrecanopathies. Morepreferably, said disease is an arthritic disease and most preferablyosteoarthritis.

In a further preferred aspect, the invention relates to a polypeptide asdescribed herein, wherein said polypeptide cross-blocks the binding toADAMTS5 of at least one of the polypeptides represented by any one ofSEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12,13, 14, 15 and 18, and/or is cross-blocked from binding to ADAMTS5 by atleast a polypeptide represented by any one of SEQ ID NO:s 2, 116, 19, 1,3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In a further preferred aspect, the invention relates to a polypeptidecross-blocking binding to ADAMTS5 by a polypeptide represented by anyone of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117,8, 12, 13, 14, 15 and 18, and/or is cross-blocked from binding toADAMTS5 by at least a polypeptide represented by any one of SEQ ID NO:s2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15and 18, wherein said polypeptide comprises at least one VH, VL, dAb,immunoglobulin single variable domain (ISVD) specifically binding toADAMTS5, wherein binding to ADAMTS5 modulates an activity of ADAMTS5.

Other aspects, advantages, applications and uses of the polypeptides andcompositions will become clear from the further disclosure herein.Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

FIGURE LEGENDS

FIG. 1: ISVDs binding ADAMTS5 do not inhibit MMP1 (A) or MMP14 (B)activity.

FIG. 2: ISVDs binding ADAMTS5 do not inhibit ADAMTS4 activity

FIG. 3: pre-existing Antibody (preAb) binding levels to Nanobodyconstruct 581 (A) and Nanobody construct 579 (B) from 3 donor samplesets derived from: healthy subjects, osteoarthritis subjects and donorswith residual preAb binding.

FIG. 4: Nanobody construct 581 (“C011400581”) does not bind to humanADAMTS1 (A), and does not bind to human ADAMTS4 or human ADAMTS15 (B).

FIG. 5: Potency in human explant system. GAG accumulation in supernatantafter 7 days is shown.

FIG. 6: A) Time course of GAG release into the supernatant in the bovineco-culture system.

-   -   B) Area under curve (AUC) calculation of GAG release over time        (7-28 days). The number of replicates n=4. Data are shown in Box        &Whiskers format with min to max.    -   C) The data show the GAG content [ng/mg cartilage] after papain        digestion. The number of replicates n=4. Data are shown in Box        &Whiskers format with min to max.

FIG. 7: Area under curve (AUC) calculation of exAGNx1 release over time(7-28 days). The number of replicates n=4. Data are shown in Box&Whiskers format with min to max.

FIG. 8: Area under curve (AUC) calculation of C2M release over time(7-28 days). The number of replicates n=4. Data are shown in Box&Whiskers format with min to max.

FIG. 9: Area under curve (AUC) calculation of C3M release over time(7-28 days). The number of replicates n=4. Data are shown in Box&Whiskers format with min to max.

FIG. 10: Inhibition of aggrecanase activity in NHP.

FIG. 11: Inhibition of cartilage degeneration. Median cartilagedegeneration sum is shown in prophylactic (A) and therapeutic (B)treatment in a DMM mouse model.

FIG. 12: Symptomatic behavior in a rat surgical OA model.

FIG. 13: Medial tibial cartilage degeneration width.

FIG. 14: The effect of the anti-ADAMTS-5 nanobody on aggrecanase derivedaggrecan degradation in ex vivo cultures of cartilage (A, B, C) andco-cultures of cartilage and synovial membrane (D, E). Theconcentrations listed are the concentration of the nanobody. Statisticalanalysis was performed with ordinary one-way ANOVA or two-way ANOVA.Statistical significance was considered, when p<0.05.

DETAILED DESCRIPTION

There remains a need for safe and efficacious OA medicaments, inparticular DMOADs. These medicaments should comply with various andfrequently opposing requirements, especially when a broadly applicableformat is intended. As such, the format should preferably be useful in abroad range of patients. The format should preferably be safe and notinduce infections due to frequent administration. In addition, theformat should preferably be patient friendly, such as a, the formatshould allowing for a convenient dosing regimen and route ofadministration, e.g. systemic administration. For instance, it ispreferred that the format is not removed instantaneously fromcirculation upon administration. However, extending the half-life shouldpreferably not introduce off-target activity and side effects or limitefficacy.

The present invention realizes at least one of these requirements.

Based on unconventional screening, characterization and combinatorystrategies, the present inventors surprisingly observed thatimmunoglobulin single variable domains (ISVDs) performed exceptionallywell in in vitro and in vivo experiments.

Moreover, the present inventors were able to re-engineer the ISVDsfurther outperforming comparator drugs in ameliorating OA. In addition,the ISVDs of the invention were also demonstrated to be significantlysafer than the prior art compounds.

The present invention intends providing polypeptides antagonizingADAMTSs in particular ADAMTS5 with improved prophylactic, therapeuticand/or pharmacological properties, including a safer profile, comparedto the prior art amino acid sequences and antibodies.

Accordingly, the present invention relates to ISVDs and polypeptidesthat are directed against/and or that may specifically bind (as definedherein) to ADAMTS5.

Accordingly, the present invention relates to ISVDs and polypeptidesthat are directed against/and or that may specifically bind (as definedherein) to ADAMTSs and modulate the activity thereof, in particular apolypeptide comprising at least one ISVD specifically binding ADAMTS5,wherein binding to ADAMTS5 modulates an activity of ADAMTS5.

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al.(Molecular Cloning: A Laboratory Manual (2^(nd) d Ed.) Vols. 1-3, ColdSpring Harbor Laboratory Press, 1989), F. Ausubel et al. (Currentprotocols in molecular biology, Green Publishing and Wiley Interscience,New York, 1987), Lewin (Genes I I, John Wiley &Sons, New York, N.Y.,1985), Old et al. (Principles of Gene Manipulation: An Introduction toGenetic Engineering (2nd edition) University of California Press,Berkeley, Calif., 1981); Roitt et al. (Immunology (6^(th) Ed.)Mosby/Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's EssentialImmunology (10^(th) Ed.) Blackwell Publishing, UK, 2001), and Janeway etal. (Immunobiology (6^(th) Ed.) Garland Science Publishing/ChurchillLivingstone, New York, 2005), as well as to the general background artcited herein.

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein; as well as to for example thefollowing reviews Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-56, 2006),Levin and Weiss (Mol. Biosyst. 2(1): 49-57, 2006), Irving et al. (J.Immunol. Methods 248(1-2): 31-45, 2001), Schmitz et al. (Placenta 21Suppl. A: S106-12, 2000), Gonzales et a. (Tumour Biol. 26(1): 31-43,2005), which describe techniques for protein engineering, such asaffinity maturation and other techniques for improving the specificityand other desired properties of proteins such as immunoglobulins.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 15%, more preferably within 10%, and most preferablywithin 5% of a given value or range.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with theterm“containing” or “including” or sometimes when used herein with theterm “having”.

The term “sequence” as used herein (for example in terms like“immunoglobulin sequence”, “antibody sequence”, “variable domainsequence”, “V_(HH) sequence” or “protein sequence”), should generally beunderstood to include both the relevant amino acid sequence as well asnucleic acids or nucleotide sequences encoding the same, unless thecontext requires a more limited interpretation.

Amino acid sequences are interpreted to mean a single amino acid or anunbranched sequence of two or more amino acids, depending of thecontext. Nucleotide sequences are interpreted to mean an unbranchedsequence of 3 or more nucleotides.

Amino acids are those L-amino acids commonly found in naturallyoccurring proteins. Amino acid residues will be indicated according tothe standard three-letter or one-letter amino acid code. Reference isfor instance made to Table A-2 on page 48 of WO 08/020079. Those aminoacid sequences containing D-amino acids are not intended to be embracedby this definition. Any amino acid sequence that containspost-translationally modified amino acids may be described as the aminoacid sequence that is initially translated using the symbols shown inthis Table A-2 with the modified positions; e.g., hydroxylations orglycosylations, but these modifications shall not be shown explicitly inthe amino acid sequence. Any peptide or protein that can be expressed asa sequence modified linkages, cross links and end caps, non-peptidylbonds, etc., is embraced by this definition, all as known in the art.

The terms “protein”, “peptide”, “protein/peptide”, and “polypeptide” areused interchangeably throughout the disclosure and each has the samemeaning for purposes of this disclosure. Each term refers to an organiccompound made of a linear chain of two or more amino acids. The compoundmay have ten or more amino acids; twenty-five or more amino acids; fiftyor more amino acids; one hundred or more amino acids, two hundred ormore amino acids, and even three hundred or more amino acids. Theskilled artisan will appreciate that polypeptides generally comprisefewer amino acids than proteins, although there is no art-recognizedcut-off point of the number of amino acids that distinguish apolypeptides and a protein; that polypeptides may be made by chemicalsynthesis or recombinant methods; and that proteins are generally madein vitro or in vivo by recombinant methods as known in the art. Byconvention, the amide bond in the primary structure of polypeptides isin the order that the amino acids are written, in which the amine end(N-terminus) of a polypeptide is always on the left, while the acid end(C-terminus) is on the right.

A nucleic acid or amino acid sequence is considered to be “(in)(essentially) isolated (form)”—for example, compared to the reactionmedium or cultivation medium from which it has been obtained—when it hasbeen separated from at least one other component with which it isusually associated in said source or medium, such as another nucleicacid, another protein/polypeptide, another biological component ormacromolecule or at least one contaminant, impurity or minor component.In particular, a nucleic acid or amino acid sequence is considered“(essentially) isolated” when it has been purified at least 2-fold, inparticular at least 10-fold, more in particular at least 100-fold, andup to 1000-fold or more. A nucleic acid or amino acid that is “in(essentially) isolated form” is preferably essentially homogeneous, asdetermined using a suitable technique, such as a suitablechromatographical technique, such as polyacrylamide-gel electrophoresis.

When a nucleotide sequence or amino acid sequence is said to “comprise”another nucleotide sequence or amino acid sequence, respectively, or to“essentially consist of” another nucleotide sequence or amino acidsequence, this may mean that the latter nucleotide sequence or aminoacid sequence has been incorporated into the first mentioned nucleotidesequence or amino acid sequence, respectively, but more usually thisgenerally means that the first mentioned nucleotide sequence or aminoacid sequence comprises within its sequence a stretch of nucleotides oramino acid residues, respectively, that has the same nucleotide sequenceor amino acid sequence, respectively, as the latter sequence,irrespective of how the first mentioned sequence has actually beengenerated or obtained (which may for example be by any suitable methoddescribed herein). By means of a non-limiting example, when apolypeptide of the invention is said to comprise an immunoglobulinsingle variable domain (“ISVD”), this may mean that said immunoglobulinsingle variable domain sequence has been incorporated into the sequenceof the polypeptide of the invention, but more usually this generallymeans that the polypeptide of the invention contains within its sequencethe sequence of the immunoglobulin single variable domains irrespectiveof how said polypeptide of the invention has been generated or obtained.Also, when a nucleic acid or nucleotide sequence is said to compriseanother nucleotide sequence, the first mentioned nucleic acid ornucleotide sequence is preferably such that, when it is expressed intoan expression product (e.g. a polypeptide), the amino acid sequenceencoded by the latter nucleotide sequence forms part of said expressionproduct (in other words, that the latter nucleotide sequence is in thesame reading frame as the first mentioned, larger nucleic acid ornucleotide sequence). Also, when a construct of the invention is said tocomprise a polypeptide or ISVD, this may mean that said construct atleast encompasses said polypeptide or ISVD, respectively, but moreusually this means that said construct encompasses groups, residues(e.g. amino acid residues), moieties and/or binding units in addition tosaid polypeptide or ISVD, irrespective of how said polypeptide or ISVDis connected to said groups, residues (e.g. amino acid residues),moieties and/or binding units and irrespective of how sad construct hasbeen generated or obtained.

By “essentially consist of” is meant that the ISVD used in the inventioneither is exactly the same as the ISVD of the invention or correspondsto an ISVD of the invention, having a limited number of amino acidresidues, such as 1-20 amino acid residues, for example 1-10 amino acidresidues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5or 6 amino acid residues, added at the amino terminal end, at thecarboxy terminal end, or at both the amino terminal end and the carboxyterminal end of the ISVD.

For the purposes of comparing two or more nucleotide sequences, thepercentage of “sequence identity” between a first nucleotide sequenceand a second nucleotide sequence may be calculated by dividing [thenumber of nucleotides in the first nucleotide sequence that areidentical to the nucleotides at the corresponding positions in thesecond nucleotide sequence] by [the total number of nucleotides in thefirst nucleotide sequence] and multiplying by [100%], in which eachdeletion, insertion, substitution or addition of a nucleotide in thesecond nucleotide sequence—compared to the first nucleotide sequence—isconsidered as a difference at a single nucleotide (position).Alternatively, the degree of sequence identity between two or morenucleotide sequences may be calculated using a known computer algorithmfor sequence alignment such as NCBI Blast v2.0, using standard settings.Some other techniques, computer algorithms and settings for determiningthe degree of sequence identity are for example described in WO04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185and GB 2357768. Usually, for the purpose of determining the percentageof “sequence identity” between two nucleotide sequences in accordancewith the calculation method outlined hereinabove, the nucleotidesequence with the greatest number of nucleotides will be taken as the“first” nucleotide sequence, and the other nucleotide sequence will betaken as the “second” nucleotide sequence.

For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence (also referred to herein as “amino acididentity”) may be calculated by dividing [the number of amino acidresidues in the first amino acid sequence that are identical to theamino acid residues at the corresponding positions in the second aminoacid sequence] by [the total number of amino acid residues in the firstamino acid sequence] and multiplying by [100%], in which each deletion,insertion, substitution or addition of an amino acid residue in thesecond amino acid sequence—compared to the first amino acid sequence—isconsidered as a difference at a single amino acid residue (position),i.e., as an “amino acid difference” as defined herein. Alternatively,the degree of sequence identity between two amino acid sequences may becalculated using a known computer algorithm, such as those mentionedabove for determining the degree of sequence identity for nucleotidesequences, again using standard settings. Usually, for the purpose ofdetermining the percentage of “sequence identity” between two amino acidsequences in accordance with the calculation method outlinedhereinabove, the amino acid sequence with the greatest number of aminoacid residues will be taken as the “first” amino acid sequence, and theother amino acid sequence will be taken as the “second” amino acidsequence.

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB 335768, WO 98/49185, WO 00/46383 and WO01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein.

Such conservative substitutions preferably are substitutions in whichone amino acid within the following groups (a)-(e) is substituted byanother amino acid residue within the same group: (a) small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides: Asp,Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg andLys; (d) large aliphatic, nonpolar residues: Met, Leu, lie, Val and Cys;and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferredconservative substitutions are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gin intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin;lie into Leu or into Val; Leu into lie or into Val; Lys into Arg, intoGin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al. (“Principles of Protein Structure”,Springer-Verlag, 1978), on the analyses of structure forming potentialsdeveloped by Chou and Fasman (Biochemistry 13: 211, 1974; Adv. Enzymol.,47: 45-149, 1978), and on the analysis of hydrophobicity patterns inproteins developed by Eisenberg et al. (Proc. Natl. Acad Sci. USA 81:140-144, 1984), Kyte and Doolittle (J. Molec. Biol. 157: 105-132, 1981),and Goldman et al. (Ann. Rev. Biophys. Chem. 15: 321-353, 1986), allincorporated herein in their entirety by reference. Information on theprimary, secondary and tertiary structure of Nanobodies is given in thedescription herein and in the general background art cited above. Also,for this purpose, the crystal structure of a V_(HH) domain from a llamais for example given by Desmyter et al. (Nature Structural Biology, 3:803, 1996), Spinelli et al. (Natural Structural Biology, 3: 752-757,1996) and Decanniere et al. (Structure, 7 (4): 361, 1999). Furtherinformation about some of the amino acid residues that in conventionalV_(H) domains form the V_(H)/V_(L) interface and potential camelizingsubstitutions on these positions can be found in the prior art citedabove.

Amino acid sequences and nucleic acid sequences are said to be “exactlythe same” if they have 100% sequence identity (as defined herein) overtheir entire length.

When comparing two amino acid sequences, the term “amino acid(s)difference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first sequence, compared to thesecond sequence; it being understood that two amino acid sequences cancontain one, two or more such amino acid differences. More particularly,in the ISVDs and/or polypeptides of the present invention, the term“amino acid(s) difference” refers to an insertion, deletion orsubstitution of a single amino acid residue on a position of the CDRsequence specified in b), d) or f), compared to the CDR sequence ofrespectively a), c) or e); it being understood that the CDR sequence ofb), d) and f) can contain one, two, three, four or maximal five suchamino acid differences compared to the CDR sequence of respectively a),c) or e).

The “amino acid(s) difference” can be any one, two, three, four ormaximal five substitutions, deletions or insertions, or any combinationthereof, that either improve the properties of the ADAMTS5 binder of theinvention, such as the polypeptide of the invention or that at least donot detract too much from the desired properties or from the balance orcombination of desired properties of the ADAMTS5 binder of theinvention, such as the polypeptide of the invention. In this respect,the resulting ADAMTS5 binder of the invention, such as the polypeptideof the invention should at least bind ADAMTS5 with the same, about thesame, or a higher affinity compared to the polypeptide comprising theone or more CDR sequences without the one, two, three, four or maximalfive substitutions, deletions or insertions. The affinity can bemeasured by any suitable method known in the art, but is preferablymeasured by a method as described in the examples section.

In this respect, the amino acid sequence of the CDRs according to b), d)and/or f) as indicated below, may be an amino acid sequence that isderived from an amino acid sequence according to a), c) and/or e)respectively by means of affinity maturation using one or moretechniques of affinity maturation known per se or as described in theExamples. For example, and depending on the host organism used so toexpress the polypeptide of the invention, such deletions and/orsubstitutions may be designed in such a way that one or more sites forpost-translational modification (such as one or more glycosylationsites) are removed, as will be within the ability of the person skilledin the art (cf. Examples).

A “Nanobody family”, “V_(HH) family” or “family” as used in the presentspecification refers to a group of Nanobodies and/or V_(HH) sequencesthat have identical lengths (i.e. they have the same number of aminoacids within their sequence) and of which the amino acid sequencebetween position 8 and position 106 (according to Kabat numbering) hasan amino acid sequence identity of 89% or more.

The terms “epitope” and “antigenic determinant”, which can be usedinterchangeably, refer to the part of a macromolecule, such as apolypeptide or protein that is recognized by antigen-binding molecules,such as immunoglobulins, conventional antibodies, immunoglobulin singlevariable domains and/or polypeptides of the invention, and moreparticularly by the antigen-binding site of said molecules. Epitopesdefine the minimum binding site for an immunoglobulin, and thusrepresent the target of specificity of an immunoglobulin.

The part of an antigen-binding molecule (such as an immunoglobulin, aconventional antibody, an immunoglobulin single variable domain and/or apolypeptide of the invention) that recognizes the epitope is called a“paratope”.

An amino acid sequence (such as an immunoglobulin single variabledomain, an antibody, a polypeptide of the invention, or generally anantigen binding protein or polypeptide or a fragment thereof) that can“bind to” or “specifically bind to”, that “has affinity for” and/or that“has specificity for” a certain epitope, antigen or protein (or for atleast one part, fragment or epitope thereof) is said to be “against” or“directed against” said epitope, antigen or protein or is a “binding”molecule with respect to such epitope, antigen or protein, or is said tobe “anti”-epitope, “anti”-antigen or “anti”-protein (e.g.,“anti”-ADAMTS5).

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as the K_(p), ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, K_(A), which equals1/K® and has units of (mol/liter)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two moleculeswill mainly be expressed in terms of the K_(D) value of theirinteraction; it being clear to the skilled person that in view of therelation K_(A)=1/K_(D), specifying the strength of molecular interactionby its K_(D) value can also be used to calculate the corresponding K_(A)value. The K_(D)-value characterizes the strength of a molecularinteraction also in a thermodynamic sense as it is related to the changeof free energy (DG) of binding by the well-known relationDG=RT.ln(K_(D)) (equivalently DG=−RT.ln(K_(A))), where R equals the gasconstant, T equals the absolute temperature and ln denotes the naturallogarithm.

The K_(D) for biological interactions which are considered meaningful(e.g. specific) are typically in the range of 10⁻¹² M (0.001 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is, the lower is its K_(D).

The K_(D) can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as k_(off), to the rate of itsassociation, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe SI unit notation of second). The on-rate k_(on) has units M⁻¹s⁻¹.The on-rate may vary between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, approachingthe diffusion-limited association rate constant for bimolecularinteractions. The off-rate is related to the half-life of a givenmolecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate may vary between 10⁻⁶ s⁻¹ (near irreversible complex with at_(1/2) of multiple days) to 1 s⁻¹ (t_(1/2)=0.69 s).

Specific binding of an antigen-binding protein, such as an ISVD, to anantigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, saturation binding assaysand/or competitive binding assays, such as radio-immunoassays (RIA),enzyme immunoassays (EIA) and sandwich competition assays, and thedifferent variants thereof known per se in the art; as well as the othertechniques mentioned herein.

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well-knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al. 2001, Intern. Immunology 13: 1551-1559) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(on),k_(off) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known BIACORE® instruments(Pharmacia Biosensor AB, Uppsala, Sweden). Kinetic Exclusion Assay(KINEXA®) (Drake et al. 2004, Analytical Biochemistry 328: 35-43)measures binding events in solution without labeling of the bindingpartners and is based upon kinetically excluding the dissociation of acomplex. In-solution affinity analysis can also be performed using theGYROLAB® immunoassay system, which provides a platform for automatedbioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis5: 1765-74), or ELISA.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artifacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition site for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules. In particular, the accuratemeasurement of K_(D) may be quite labor-intensive and as a consequence,often apparent K_(D) values are determined to assess the bindingstrength of two molecules. It should be noted that as long as allmeasurements are made in a consistent way (e.g. keeping the assayconditions unchanged) apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule or antigen-binding protein (such as an ISVD or polypeptide ofthe invention) molecule can bind. The specificity of an antigen-bindingprotein can be determined based on affinity and/or avidity, for instanceas described on pages 53-56 of WO 08/020079 (incorporated herein byreference), which also describes some preferred techniques for measuringbinding between an antigen-binding molecule (such as a polypeptide orISVD of the invention) and the pertinent antigen. Typically,antigen-binding proteins (such as the ISVDs and/or polypeptides of theinvention) will bind to their antigen with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter(i.e., with an association constant (K_(A)) of 10⁵ to 10¹² liter/molesor more, and preferably 10⁷ to 10¹² liter/moles or more and morepreferably 10⁸ to 10¹² liter/moles). Any K_(D) value greater than 10⁻⁴mol/liter (or any K_(A) value lower than 10⁴ liter/mol) is generallyconsidered to indicate non-specific binding. Preferably, a monovalentISVD of the invention will bind to the desired antigen with an affinityless than 500 nM, preferably less than 200 nM, more preferably less than10 nM, such as less than 500 pM, such as e.g., between 10 and 5 pM orless. Reference is also made to paragraph n) on pages 53-56 of WO08/020079.

An ISVD and/or polypeptide is said to be “specific for” a (first) targetor antigen compared to another (second) target or antigen when it bindsto the first antigen with an affinity (as described above, and suitablyexpressed as a K_(D) value, K_(A) value, K_(off) rate and/or K_(on)rate) that is at least 10 times, such as at least 100 times, andpreferably at least 1000 times or more better than the affinity withwhich the ISVD and/or polypeptide binds to the second target or antigen.For example, the ISVD and/or polypeptide may bind to the first target orantigen with a K₀ value that is at least 10 times less, such as at least100 times less, and preferably at least 1000 times less or even lessthan that, than the K_(D) with which said ISVD and/or polypeptide bindsto the second target or antigen. Preferably, when an ISVD and/orpolypeptide is “specific for” a first target or antigen compared to asecond target or antigen, it is directed against (as defined herein)said first target or antigen, but not directed against said secondtarget or antigen.

Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, saturation binding assays and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and the different variants thereof known in the art; as well asthe other techniques mentioned herein.

A preferred approach that may be used to assess affinity is the 2-stepELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al.1985 (J. Immunol. Methods 77: 305-19). This method establishes asolution phase binding equilibrium measurement and avoids possibleartifacts relating to adsorption of one of the molecules on a supportsuch as plastic. As will be clear to the skilled person, thedissociation constant may be the actual or apparent dissociationconstant. Methods for determining the dissociation constant will beclear to the skilled person, and for example include the techniquesmentioned on pages 53-56 of WO 08/020079.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or FACS (Fluorescent activatedcell sorting) or other format (the fluorophore for fluorescencedetection, the chromophore for light absorption detection, the biotinfor streptavidin-mediated ELISA detection). Typically, the referencemolecule C is kept at a fixed concentration and the concentration of Ais varied for a given concentration or amount of B. As a result an IC₅₀value is obtained corresponding to the concentration of A at which thesignal measured for C in absence of A is halved. Provided K_(D ref), theK_(D) of the reference molecule, is known, as well as the totalconcentration c_(ref) of the reference molecule, the apparent K_(D) forthe interaction A-B can be obtained from following formula:K_(D)=IC₅₀/(1+c_(ref)/K_(Dref)). Note that if c_(ref)<<K_(D ref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the difference in strength or stability of a molecularinteraction can be assessed by comparing the IC₅₀ and this measurementis judged as equivalent to K_(D) or to apparent K_(D) throughout thistext.

The half maximal inhibitory concentration (IC₅₀) can also be a measureof the effectiveness of a compound in inhibiting a biological orbiochemical function, e.g. a pharmacological effect. This quantitativemeasure indicates how much of the polypeptide or ISVD (e.g. a Nanobody)is needed to inhibit a given biological process (or component of aprocess, i.e. an enzyme, cell, cell receptor, chemotaxis, anaplasia,metastasis, invasiveness, etc.) by half. In other words, it is the halfmaximal (50%) inhibitory concentration (IC) of a substance (50% IC, orIC₅₀). IC₅₀ values can be calculated for a given antagonist such as thepolypeptide or ISVD (e.g. a Nanobody) of the invention by determiningthe concentration needed to inhibit half of the maximum biologicalresponse of the agonist. The K_(p) of a drug can be determined byconstructing a dose-response curve and examining the effect of differentconcentrations of antagonist such as the polypeptide or ISVD (e.g. aNanobody) of the invention on reversing agonist activity.

The term half maximal effective concentration (EC₅₀) refers to theconcentration of a compound which induces a response halfway between thebaseline and maximum after a specified exposure time. In the presentcontext it is used as a measure of a polypeptide, ISVD (e.g. a Nanobody)its potency. The EC₅₀ of a graded dose response curve represents theconcentration of a compound where 50% of its maximal effect is observed.Concentration is preferably expressed in molar units.

In biological systems, small changes in ligand concentration typicallyresult in rapid changes in response, following a sigmoidal function. Theinflection point at which the increase in response with increasingligand concentration begins to slow is the EC₅₀. This can be determinedmathematically by derivation of the best-fit line. Relying on a graphfor estimation is convenient in most cases. In case the EC₅₀ is providedin the examples section, the experiments were designed to reflect theK_(D) as accurate as possible. In other words, the EC₅₀ values may thenbe considered as K_(D) values. The term “average K_(D)” relates to theaverage K_(D) value obtained in at least 1, but preferably more than 1,such as at least 2 experiments. The term “average” refers to themathematical term “average” (sums of data divided by the number of itemsin the data).

It is also related to IC₅₀ which is a measure of a compound itsinhibition (50% inhibition). For competition binding assays andfunctional antagonist assays IC₅₀ is the most common summary measure ofthe dose-response curve. For agonist/stimulator assays the most commonsummary measure is the EC₅₀.

The inhibition constant (Ki) is an indication of how potent an inhibitoris; it is the concentration required to produce half maximum inhibition.Unlike IC₅₀, which can change depending on the experimental conditions,Ki is an absolute value and is often referred to as the inhibitionconstant of a drug. The inhibition constant K_(i) can be calculated byusing the Cheng-Prusoff equation:

$K_{i} = \frac{{IC}\; 50}{\frac{\lbrack L\rbrack}{K_{D}} + 1}$

in which [L] is the fixed concentration of the ligand.

The term “potency” of a polypeptide and/or ISVD of the invention, asused herein, is a function of the amount of polypeptide and/or ISVD ofthe invention required for its specific effect to occur. It refers tothe capacity of said polypeptide and/or ISVD of the invention tomodulate and/or partially or fully inhibit an activity of ADAMTS5. Moreparticularly, it may refer to the capacity of said polypeptide and/orISVD to reduce or even totally inhibit an ADAMTS5 activity as definedherein. As such, it may refer to the capacity of said polypeptide and/orISVD to inhibit an activity of ADAMTS5, such as an enzymatic activity,such as proteolysis, e.g. the protease activity and/or endopeptidaseactivities, as well as binding of a substrate, including but not limitedto Aggrecan, versican, brevican, neurocan, decorin, and/or biglycan,preferably cleavage of Aggrecan. Said polypeptide and/or ISVD preferablyantagonizes aggrecanase activity of ADAMTS5. The potency may be measuredby any suitable assay known in the art or described herein. As usedherein, “aggrecanase activity” is defined as the proteolytic cleavage ofAggrecan.

The “efficacy” of the polypeptide of the invention measures the maximumstrength of the effect itself, at saturating polypeptide concentrations.Efficacy indicates the maximum response achievable from the polypeptideof the invention. It refers to the ability of a polypeptide to producethe desired (therapeutic) effect.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide binds to ADAMTS5 with a K_(D) between 1 E⁻⁰⁷ Mand 1 E⁻¹³ M, such as between 1 E⁻⁰⁸ M and 1 E⁻¹² M, preferably at most1 E⁻⁰⁷ M, preferably lower than 1 E⁻⁰⁸ M or 1 E⁻⁰⁹ M, or even lower than1 E⁻¹⁰ M, such as 5 E⁻¹M, 4 E⁻¹¹ M, 3 E⁻¹¹ M, 2 E⁻¹¹ M, 1.7 E⁻¹¹ M, 1E⁻¹¹ M, or even 5 E⁻¹² M, 4 E⁻¹² M, 3 E⁻¹² M, 1 E⁻¹² M, for instance asdetermined by Gyrolab or KinExA.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide modulates ADAMTS5 with an EC₅₀ between 1 E⁻⁰⁷ Mand 1 E⁻¹² M, such as between 1 E⁻⁰⁸ M and 1 E⁻¹¹ M, for instance asdetermined by binding ELISA.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide binds to ADAMTS5 with an off-rate of less than5 E⁻⁰⁴ s⁻¹, such as less than 1 E⁻⁰⁴ s⁻¹ or 5 E⁻⁰⁵ s⁻¹, or even lessthan 1 E⁻⁰⁵ s⁻¹, for instance as determined by SPR.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide inhibits an activity of ADAMTS5 with an IC₅₀between 1 E⁻⁰⁷ M and 1 E⁻¹² M, such as between 1 E⁻⁰⁸ M and 1 E⁻¹¹ M,for instance as determined by human FRET assay or human AlphaLISA.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide inhibits an enzymatic activity of ADAMTS5 withan IC₅₀ of at most 1 E⁻⁰⁷ M, preferably 1 E⁻⁰⁸ M, 5 E⁻⁰⁹ M, or 4 E⁻⁹ M,3 E⁻⁹ M, 2 E⁻⁹ M, such as 1 E⁻⁹ M.

An amino acid sequence, such as an ISVD or polypeptide, is said to be“cross-reactive” for two different antigens or antigenic determinants(such as e.g., ADAMTS5 from different species of mammal, such as e.g.,human ADAMTS5, bovine ADAMTS5, rat ADAMTS5, guinea pig ADAMTS5, mouseADAMTS5 or cynomolgus ADAMTS5) if it is specific for (as defined herein)these different antigens or antigenic determinants. It will beappreciated that an ISVD or polypeptide may be considered to becross-reactive although the binding affinity for the two differentantigens can differ, such as by a factor, 2, 5, 10, 50, 100 or even moreprovided it is specific for (as defined herein) these different antigensor antigenic determinants.

ADAMTS5 is also known as ADAMTS11, ADMP-2 or Aggrecanase-2.

Relevant structural information for ADAMTS5 may be found, for example,at UniProt Accession Numbers as depicted in the Table 1 below (cf. TableB).

TABLE 1 Protein Acc. Gene Organism SEQ ID NO: Q9UNA0 ADAMTS5 H. sapiens149 Q9TT92 ADAMTS5 B. taurus 150 Q6TY19 ADAMTS5 R. norvegicus 151 H0VFP0ADAMTS5 Cavia Porcellus 152 Q9R001 ADAMTS5 M. musculus 153 F6Z3S6ADAMTS5 M. mulatta 154

“Human ADAMTS5” refers to the ADAMTS5 comprising the amino acid sequenceof SEQ ID NO: 149. In an aspect the polypeptide of the inventionspecifically binds ADAMTS5 from Human sapiens, Mus musculus, CaviaPorcellus, Bos taurus, Macaca mulatta and/or Rattus norvegicus,preferably human ADAMTS5, preferably SEQ ID NO: 149.

The terms “(cross)-block”, “(cross)-blocked”, “(cross)-blocking”,“competitive binding”, “(cross)-compete”, “(cross)-competing” and“(cross)-competition” are used interchangeably herein to mean theability of an immunoglobulin, antibody, ISVD, polypeptide or otherbinding agent to interfere with the binding of other immunoglobulins,antibodies, ISVDs, polypeptides or binding agents to a given target. Theextent to which an immunoglobulin, antibody, ISVD, polypeptide or otherbinding agent is able to interfere with the binding of another to thetarget, and therefore whether it may be said to cross-block according tothe invention, may be determined using competition binding assays, whichare common in the art, such as, for instance, by screening purifiedISVDs against ISVDs displayed on phage in a competition ELISA.Particularly suitable quantitative cross-blocking assays include ELISA.

Other methods for determining whether an immunoglobulin, antibody, ISVD,polypeptide or other binding agent directed against a target(cross)-blocks, is capable of (cross)-blocking, competitively binds oris (cross)-competitive as defined herein, can be evaluated by anSPR-based “sandwich assay”, such as for instance described in theExamples section. Other suitable methods are described e.g. in Xiao-ChiJia et al. (Journal of Immunological Methods 288: 91-98, 2004), Milleret al. (Journal of Immunological Methods 365: 118-125, 2011).

Accordingly, the present invention relates to a polypeptide as describedherein, such as represented by SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17,10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18 (cf. Table A-1),wherein said polypeptide competes with a cross-blocking polypeptide, forinstance as determined by competition ELISA.

The present invention relates to a method for determining competitors,such as polypeptides, competing with a polypeptide as described herein,such as represented by any one of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16,17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18, wherein thepolypeptide as described herein competes with or cross blocks thecompetitor, such as a polypeptide, for binding to ADAMTS5, such as, forinstance human ADAMTS5 (SEQ ID NO: 149), wherein the binding to ADAMTS5of the competitor is reduced by at least 5%, such as 10%, 20%, 30%, 40%,50% or even more, such as 80%, 90% or even 100% (i.e. virtuallyundetectable in a given assay) in the presence of a polypeptide of theinvention, compared to the binding to ADAMTS5 of the competitor in theabsence of the polypeptide of the invention. Competition and crossblocking may be determined by any means known in the art, such as, forinstance, competition ELISA or FACS assay. In an aspect the presentinvention relates to a polypeptide of the invention, wherein saidpolypeptide cross-blocks the binding to ADAMTS5 of at least one of thepolypeptides represented by SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10,11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18 and/or is cross-blockedfrom binding to ADAMTS5 by at least one of the polypeptides representedby SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8,12, 13, 14, or 18.

The present invention also relates to competitors competing with apolypeptide as described herein, such as SEQ ID NO:s 2, 116, 19, 1, 3,6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 or 18, wherein thecompetitor competes with or cross blocks the polypeptide as describedherein for binding to ADAMTS5, wherein the binding to ADAMTS5 of thepolypeptide of the invention is reduced by at least 5%, such as 10%,20%, 30%, 40%, 50% or even more, such as 80%, or even more such as atleast 90% or even 100% (i.e. virtually undetectable in a given assay) inthe presence of said competitor, compared to the binding to ADAMTS5 bythe polypeptide of the invention in the absence of said competitor. Inan aspect the present invention relates to a polypeptide cross-blockingbinding to ADAMTS5 by a polypeptide of the invention such as one of SEQID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13,14, 15 or 18 and/or is cross-blocked from binding to ADAMTS5 by at leastone of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117,8, 12, 13, 14, 15 or 18, preferably wherein said polypeptide comprisesat least one VH, VL, dAb, or ISVD specifically binding to ADAMTS5,wherein binding to ADAMTS5 modulates an activity of ADAMTS5.

“ADAMTS5 activities” and “activities of ADAMTS5” (these terms are usedinterchangeably herein) include, but are not limited to enzymaticactivities, such as proteolysis, e.g. protease activity (also calledproteinase or peptidase activity), and endopeptidase activities, on theone hand, and the activities by the exosites, such as for instancerecognizing and/or binding the substrate, e.g. by disintegrin-likedomain, central thrombospondin type I-like (TS) repeat, cysteine-richdomain, spacer region and/or additional TS motifs. ADAMTS5 activitiesinclude binding and/or proteolysis of substrates such ashyaluronan-binding chondroitin sulfate proteoglycan (CSPG) extracellularproteins, such as Aggrecan, versican, brevican, neurocan, decorin andbiglycan. As used herein, proteolysis is the breakdown of proteins intosmaller polypeptides or amino acids by hydrolysis of the peptide bondsthat link amino acids together in a polypeptide chain.

In the context of the present invention, “modulating” or “to modulate”generally means altering an activity by ADAMTS5, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein). In particular, “modulating” or “to modulate” may mean eitherreducing or inhibiting an activity of, or alternatively increasing anactivity of ADAMTS5, as measured using a suitable in vitro, cellular orin vivo assay (for instance, such as those mentioned herein), by atleast 1%, preferably at least 5%, such as at least 10% or at least 25%,for example by at least 50%, at least 60%, at least 70%, at least 80%,or 90% or more, compared to activity of ADAMTS5 in the same assay underthe same conditions but without the presence of the ISVD or polypeptideof the invention.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide modulates an activity of ADAMTS5,preferably inhibiting an activity of ADAMTS5.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide inhibits protease activity of ADAMTS5,such as inhibits the proteolysis of a substrate, such as Aggrecan,versican, brevican, neurocan, decorin, and/or biglycan.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide blocks the binding of ADAMTS5 to asubstrate, such as Aggrecan, versican, brevican, neurocan, decorin,and/or biglycan, wherein said substrate is preferably Aggrecan.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide blocks the binding of ADAMTS5 to Aggrecan of atleast 20%, such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% oreven more, for instance as determined by ELISA.

In an aspect the invention relates to a polypeptide as described herein,wherein said polypeptide antagonizes or inhibits an activity of ADAMTS5,such as (i) a protease activity, preferably cleavage of Aggrecan,versican, brevican, neurocan, decorin, and/or biglycan, preferablycleavage of Aggrecan; preferably antagonizes aggrecanase activity ofADAMTS5; (ii) binding of a substrate to ADAMTS5, such as an exosite ofADAMTS5, for instance the disintegrin-like domain, the centralthrombospondin type I-like (TS) repeat, the cysteine-rich domain, thespacer region or the additional TS motif.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said polypeptide inhibits protease activity of ADAMTS5,preferably by at least 5%, such as 10%, 20%, 30%, 40%, 50% or even more,such as at least 60%, 70%, 80%, 90%, 95% or even more, as determined byany suitable method known in the art, such as for instance by enzymeinhibition assays or as described in the Examples section.

Although the ADAMs, ADAMTSs and MMPs share a binding site to Aggrecanthat is very similar both in sequence and in overall shape, e.g., thecatalytic domains of ADAMTS4 and ADAMTS5 share a high degree of sequencesimilarity, the inventors were able to identify ISVDs which were targetspecific, as demonstrated in the examples. The target specificity alsowould avoid or at least limit musculoskeletal syndrome, which is aside-effect caused by broad-spectrum inhibitors.

In an aspect the invention relates to an ADAMTS5 binder such as an ISVDand polypeptide of the invention, wherein said ADAMTS5 binder does notbind ADAMTS4, ADAMTS1, ADAMTS15, MMP1 and/or MMP14 (membrane type).Preferably, the present invention relates to a polypeptide as definedherein, wherein said ISVD binding ADAMTS5 does not bind ADAMTS4, MMP1 orMMP14.

Unless indicated otherwise, the terms “immunoglobulin” and“immunoglobulin sequence”—whether used herein to refer to a heavy chainantibody or to a conventional 4-chain antibody—is used as a general termto include both the full-size antibody, the individual chains thereof,as well as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as V_(HH) domainsor V_(H)/V_(L) domains, respectively).

The term “domain” (of a polypeptide or protein) as used herein refers toa folded protein structure which has the ability to retain its tertiarystructure independently of the rest of the protein. Generally, domainsare responsible for discrete functional properties of proteins, and inmany cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g., a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin foldcharacteristic of antibody molecules, which consists of a two-layersandwich of about seven antiparallel beta-strands arranged in twobeta-sheets, optionally stabilized by a conserved disulphide bond.

The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and herein below as “framework region1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3”or “FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementarity determiningregions” or “CDRs”, which are referred to in the art and herein below as“complementarity determining region 1” or “CDR1”; as “complementaritydetermining region 2” or “CDR2”; and as “complementarity determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain may be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site.

The term “immunoglobulin single variable domain” (abbreviated herein as“ISVD” or “ISV”), and interchangeably used with “single variabledomain”, defines molecules wherein the antigen binding site is presenton, and formed by, a single immunoglobulin domain. This setsimmunoglobulin single variable domains apart from “conventional”immunoglobulins or their fragments, wherein two immunoglobulin domains,in particular two variable domains, interact to form an antigen bindingsite. Typically, in conventional immunoglobulins, a heavy chain variabledomain (V_(H)) and a light chain variable domain (V₁) interact to forman antigen binding site. In the latter case, the complementaritydetermining regions (CDRs) of both V_(H) and V_(L) will contribute tothe antigen binding site, i.e. a total of 6 CDRs will be involved inantigen binding site formation.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e., by a V_(H)-V_(L) pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

In contrast, ISVDs are capable of specifically binding to an epitope ofthe antigen without pairing with an additional immunoglobulin variabledomain. The binding site of an ISVD is formed by a single V_(HH), V_(H)or V_(L) domain. Hence, the antigen binding site of an ISVD is formed byno more than three CDRs.

As such, the single variable domain may be a light chain variable domainsequence (e.g., a V_(L)-sequence) or a suitable fragment thereof; or aheavy chain variable domain sequence (e.g., a V_(H)-sequence or V_(HH)sequence) or a suitable fragment thereof; as long as it is capable offorming a single antigen binding unit (i.e., a functional antigenbinding unit that essentially consists of the single variable domain,such that the single antigen binding domain does not need to interactwith another variable domain to form a functional antigen binding unit).

In one embodiment of the invention, the ISVDs are heavy chain variabledomain sequences (e.g., a V_(H)-sequence); more specifically, the ISVDsmay be heavy chain variable domain sequences that are derived from aconventional four-chain antibody or heavy chain variable domainsequences that are derived from a heavy chain antibody.

For example, the ISVD may be a (single) domain antibody (or an aminoacid that is suitable for use as a (single) domain antibody), a “dAb” ordAb (or an amino acid that is suitable for use as a dAb) or a Nanobody(as defined herein, and including but not limited to a VHH); othersingle variable domains, or any suitable fragment of any one thereof.

In particular, the ISVD may be a Nanobody® (as defined herein) or asuitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone®are registered trademarks of Ablynx N.V.] For a general description ofNanobodies, reference is made to the further description below, as wellas to the prior art cited herein, such as e.g. described in WO 08/020079(page 16).

“V_(HH) domains”, also known as VHHs, V_(H)H domains, VHH antibodyfragments, and VHH antibodies, have originally been described as theantigen binding immunoglobulin (variable) domain of “heavy chainantibodies” (i.e., of “antibodies devoid of light chains”;Hamers-Casterman et al. 1993 Nature 363: 446-448). The term “V_(HH)domain” has been chosen in order to distinguish these variable domainsfrom the heavy chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “V_(H) domains” or“VH domains”) and from the light chain variable domains that are presentin conventional 4-chain antibodies (which are referred to herein as“V_(L) domains” or “VL domains”). For a further description of VHH's andNanobodies, reference is made to the review article by Muyldermans(Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as tothe following patent applications, which are mentioned as generalbackground art: WO 94/04678, WO 95/04079 and WO 96/34103 of the VrijeUniversiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie(VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 bythe National Research Council of Canada; WO 03/025020 (=EP 1433793) bythe Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V.and the further published patent applications by Ablynx N.V. Referenceis also made to the further prior art mentioned in these applications,and in particular to the list of references mentioned on pages 41-43 ofthe International application WO 06/040153, which list and referencesare incorporated herein by reference. As described in these references,Nanobodies (in particular VHH sequences and partially humanizedNanobodies) can in particular be characterized by the presence of one ormore “Hallmark residues” in one or more of the framework sequences. Afurther description of the Nanobodies, including humanization and/orcamelization of Nanobodies, as well as other modifications, parts orfragments, derivatives or “Nanobody fusions”, multivalent constructs(including some non-limiting examples of linker sequences) and differentmodifications to increase the half-life of the Nanobodies and theirpreparations may be found e.g. in WO 08/101985 and WO 08/142164. For afurther general description of Nanobodies, reference is made to theprior art cited herein, such as e.g. described in WO 08/020079 (page16).

In particular, the framework sequences present in the ADAMTS5 binders ofthe invention, such as the ISVDs and/or polypeptides of the invention,may contain one or more of Hallmark residues (for instance as describedin WO 08/020079 (Tables A-3 to A-8)), such that the ADAMTS5 binder ofthe invention is a Nanobody. Some preferred, but non-limiting examplesof (suitable combinations of) such framework sequences will become clearfrom the further disclosure herein (see e.g., Table A-2). Generally,Nanobodies (in particular V_(HH) sequences and partially humanizedNanobodies) can in particular be characterized by the presence of one ormore “Hallmark residues” in one or more of the framework sequences (ase.g., further described in WO 08/020079, page 61, line 24 to page 98,line 3).

More in particular, the invention provides ADAMTS5 binders comprising atleast one immunoglobulin single variable domain that is an amino acidsequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and        which:

-   i) have at least 80%, more preferably 90%, even more preferably 95%    amino acid identity with at least one of the amino acid sequences of    SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8,    12, 13, 14, 15 or 18 (see Table A-1), in which for the purposes of    determining the degree of amino acid identity, the amino acid    residues that form the CDR sequences are disregarded. In this    respect, reference is also made to Table A-2, which lists the    framework 1 sequences (SEQ ID NOs: 72-84), framework 2 sequences    (SEQ ID NOs: 85-94), framework 3 sequences (SEQ ID NOs: 95-113) and    framework 4 sequences (SEQ ID NOs: 114-115) of the immunoglobulin    single variable domains of SEQ ID NOs: 2, 116, 19, 1, 3, 6, 16, 17,    10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18; or

-   ii) combinations of framework sequences as depicted in Table A-2;    and in which:

-   iii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues such as mentioned in    Table A-3 to Table A-8 of WO 08/020079.

The ADAMTS5 binders of the invention, such as the ISVDs and/orpolypeptides of the invention, may also contain the specificmutations/amino acid residues described in the following co-pending USprovisional applications, all entitled “Improved immunoglobulin variabledomains”: U.S. 61/994,552 filed May 16, 2014; U.S. 61/014,015 filed Jun.18, 2014; U.S. 62/040,167 filed Aug. 21, 2014; and U.S. 62/047,560,filed Sep. 8, 2014 (all assigned to Ablynx N.V.).

In particular, the ADAMTS5 binders of the invention, such as the ISVDsand/or polypeptides of the invention, may suitably contain (i) a K or Qat position 112; or (ii) a K or Q at position 110 in combination with aV at position 11; or (iii) a T at position 89; or (iv) an L on position89 with a K or Q at position 110; or (v) a V at position 11 and an L atposition 89; or any suitable combination of (i) to (v).

As also described in said co-pending US provisional applications, whenthe ADAMTS5 binder of the invention, such as the ISVD and/or polypeptideof the invention, contain the mutations according to one of (i) to (v)above (or a suitable combination thereof):

-   -   the amino acid residue at position 11 is preferably chosen from        L, V or K (and is most preferably V); and/or    -   the amino acid residue at position 14 is preferably suitably        chosen from A or P; and/or    -   the amino acid residue at position 41 is preferably suitably        chosen from A or P; and/or    -   the amino acid residue at position 89 is preferably suitably        chosen from T, V or L; and/or    -   the amino acid residue at position 108 is preferably suitably        chosen from Q or L; and/or    -   the amino acid residue at position 110 is preferably suitably        chosen from T, K or Q; and/or    -   the amino acid residue at position 112 is preferably suitably        chosen from S, K or Q.

As mentioned in said co-pending US provisional applications, saidmutations are effective in preventing or reducing binding of so-called“pre-existing antibodies” to the immunoglobulins and compounds of theinvention. For this purpose, the ADAMTS5 binders of the invention, suchas the ISVDs and/or polypeptides of the invention, may also contain(optionally in combination with said mutations) a C-terminal extension(X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5(and preferably 1 or 2, such as 1); and each X is an (preferablynaturally occurring) amino acid residue that is so independently chosen,and preferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (1)), see e.g.US provisional applications as well as WO 12/175741. In particular, anADAMTS5 binder of the invention, such as an ISVD and/or polypeptide ofthe invention, may contain such a C-terminal extension when it forms theC-terminal end of a protein, polypeptide or other compound or constructcomprising the same (see e.g. said US provisional applications as wellas WO 12/175741).

An ADAMTS5 binder of the invention may be an immunoglobulin, such as animmunoglobulin single variable domain, derived in any suitable mannerand from any suitable source, and may for example be naturally occurringV_(HH) sequences (i.e., from a suitable species of Camelid) or syntheticor semi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies or VHH sequences, “camelized”(as defined herein) immunoglobulin sequences (and in particularcamelized heavy chain variable domain sequences), as well as Nanobodiesthat have been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing as further describedherein. Also, when an immunoglobulin comprises a V_(HH) sequence, saidimmunoglobulin may be suitably humanized, as further described herein,so as to provide one or more further (partially or fully) humanizedimmunoglobulins of the invention. Similarly, when an immunoglobulincomprises a synthetic or semi-synthetic sequence (such as a partiallyhumanized sequence), said immunoglobulin may optionally be furthersuitably humanized, again as described herein, again so as to provideone or more further (partially or fully) humanized immunoglobulins ofthe invention.

“Domain antibodies”, also known as “Dab”s, “Domain Antibodies”, and“dAbs” (the terms “Domain Antibodies” and “dAbs” being used astrademarks by the GlaxoSmithKline group of companies) have beendescribed in e.g., EP 0368684, Ward et al. (Nature 341: 544-546, 1989),Holt et a. (Tends in Biotechnology 21: 484-490, 2003) and WO 03/002609as well as for example WO 04/068820, WO 06/030220, WO 06/003388 andother published patent applications of Domantis Ltd. Domain antibodiesessentially correspond to the VH or VL domains of non-camelidmammalians, in particular human 4-chain antibodies. In order to bind anepitope as a single antigen binding domain, i.e., without being pairedwith a VL or VH domain, respectively, specific selection for suchantigen binding properties is required, e.g. by using libraries of humansingle VH or VL domain sequences. Domain antibodies have, like VHHs, amolecular weight of approximately 13 to approximately 16 kDa and, ifderived from fully human sequences, do not require humanization for e.g.therapeutical use in humans.

It should also be noted that, although less preferred in the context ofthe present invention because they are not of mammalian origin, singlevariable domains can be derived from certain species of shark (forexample, the so-called “IgNAR domains”, see for example WO 05/18629).

The present invention relates particularly to ISVDs, wherein said ISVDsare chosen from the group consisting of VHHs, humanized VHHs andcamelized VHs.

The amino acid residues of a VHH domain are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to VHH domains fromCamelids, as shown e.g., in FIG. 2 of Riechmann and Muyldermans (J.Immunol. Methods 231: 25-38, 1999), all as known in the art. Alternativemethods for numbering the amino acid residues of V_(H) domains, whichmethods can also be applied in an analogous manner to VHH domains, areknown in the art. However, in the present description, claims andfigures, the numbering according to Kabat applied to VHH domains asdescribed above will be followed, unless indicated otherwise.

It should be noted that—as is well known in the art for V_(H) domainsand for VHH domains—the total number of amino acid residues in each ofthe CDRs may vary and may not correspond to the total number of aminoacid residues indicated by the Kabat numbering (that is, one or morepositions according to the Kabat numbering may not be occupied in theactual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence. The total number of amino acid residues in a VH domainand a VHH domain will usually be in the range of from 110 to 120, oftenbetween 112 and 115. It should however be noted that smaller and longersequences may also be suitable for the purposes described herein.

With regard to the CDRs, as is well-known in the art, there are multipleconventions to define and describe the CDRs of a VH or VHH fragment,such as the Kabat definition (which is based on sequence variability andis the most commonly used) and the Chothia definition (which is based onthe location of the structural loop regions). Reference is for examplemade to the website http://www.bioinf.org.uk/abs/. For the purposes ofthe present specification and claims the CDRs are most preferablydefined on the basis of the Abm definition (which is based on OxfordMolecular's AbM antibody modelling software), as this is considered tobe an optimal compromise between the Kabat and Chothia definitions (cf.http://www.bioinf.org.uk/abs/). As used herein, FR1 comprises the aminoacid residues at positions 1-25, CDR1 comprises the amino acid residuesat positions 26-35, FR2 comprises the amino acids at positions 36-49,CDR2 comprises the amino acid residues at positions 50-58, FR3 comprisesthe amino acid residues at positions 59-94, CDR3 comprises the aminoacid residues at positions 95-102, and FR4 comprises the amino acidresidues at positions 103-113.

In the meaning of the present invention, the term “immunoglobulin singlevariable domain” or “single variable domain” comprises polypeptideswhich are derived from a non-human source, preferably a camelid,preferably a camelid heavy chain antibody. They may be humanized, asdescribed herein. Moreover, the term comprises polypeptides derived fromnon-camelid sources, e.g. mouse or human, which have been “camelized”,as described herein.

Hence, ISVDs such as Domain antibodies and Nanobodies (including VHHdomains) may be subjected to humanization. In particular, humanizedISVDs, such as Nanobodies (including VHH domains) may be ISVDs that areas generally defined herein, but in which at least one amino acidresidue is present (and in particular, in at least one of the frameworkresidues) that is and/or that corresponds to a humanizing substitution(as defined herein). Potentially useful humanizing substitutions may beascertained by comparing the sequence of the framework regions of anaturally occurring V_(HH) sequence with the corresponding frameworksequence of one or more closely related human V_(H) sequences, afterwhich one or more of the potentially useful humanizing substitutions (orcombinations thereof) thus determined may be introduced into said V_(HH)sequence (in any manner known per se, as further described herein) andthe resulting humanized V_(HH) sequences may be tested for affinity forthe target, for stability, for ease and level of expression, and/or forother desired properties. In this way, by means of a limited degree oftrial and error, other suitable humanizing substitutions (or suitablecombinations thereof) may be determined by the skilled person based onthe disclosure herein. Also, based on the foregoing, (the frameworkregions of) an ISVD, such as a Nanobody (including VHH domains) may bepartially humanized or fully humanized.

Another particularly preferred class of ISVDs of the invention comprisesISVDs with an amino acid sequence that corresponds to the amino acidsequence of a naturally occurring V_(H) domain, but that has been“camelized”, i.e. by replacing one or more amino acid residues in theamino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe description herein. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theV_(H)—V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see also for example WO 94/04678 and Daviesand Riechmann (1994 and 1996)). Preferably, the V_(H) sequence that isused as a starting material or starting point for generating ordesigning the camelized immunoglobulin single variable domains ispreferably a V_(H) sequence from a mammal, more preferably the V_(H)sequence of a human being, such as a V_(H)3 sequence. However, it shouldbe noted that such camelized immunoglobulin single variable domains ofthe invention can be obtained in any suitable manner known per se andthus are not strictly limited to polypeptides that have been obtainedusing a polypeptide that comprises a naturally occurring V_(H) domain asa starting material. Reference is made to Davies and Riechmann (FEBS339: 285-290, 1994; Biotechnol. 13: 475-479, 1995; Prot. Eng. 9:531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231:25-38, 1999)

For example, again as further described herein, both “humanization” and“camelization” can be performed by providing a nucleotide sequence thatencodes a naturally occurring V_(HH) domain or V_(H) domain,respectively, and then changing, in a manner known per se, one or morecodons in said nucleotide sequence in such a way that the new nucleotidesequence encodes a “humanized” or “camelized” ISVD of the invention,respectively. This nucleic acid can then be expressed in a manner knownper se, so as to provide the desired ISVDs of the invention.Alternatively, based on the amino acid sequence of a naturally occurringV_(HH) domain or V_(H) domain, respectively, the amino acid sequence ofthe desired humanized or camelized ISVDs of the invention, respectively,can be designed and then synthesized de novo using techniques forpeptide synthesis known per se. Also, based on the amino acid sequenceor nucleotide sequence of a naturally occurring V_(HH) domain or V_(H)domain, respectively, a nucleotide sequence encoding the desiredhumanized or camelized ISVDs of the invention, respectively, can bedesigned and then synthesized de novo using techniques for nucleic acidsynthesis known per se, after which the nucleic acid thus obtained canbe expressed in a manner known per se, so as to provide the desiredISVDs of the invention.

ISVDs such as Domain antibodies and Nanobodies (including VHH domainsand humanized VHH domains), can also be subjected to affinity maturationby introducing one or more alterations in the amino acid sequence of oneor more CDRs, which alterations result in an improved affinity of the soresulting ISVD for its respective antigen, as compared to the respectiveparent molecule. Affinity-matured ISVD molecules of the invention may beprepared by methods known in the art, for example, as described by Markset al. (Biotechnology 10:779-783, 1992), Barbas, et al. (Proc. Nat.Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147-155,1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J.Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889 896,1992), Johnson and Hawkins (Affinity maturation of antibodies usingphage display, Oxford University Press, 1996).

The process of designing/selecting and/or preparing a polypeptide,starting from an ISVD such as an, V_(H), V_(L), V_(HH), Domain antibodyor a Nanobody, is also referred to herein as “formatting” said ISVD; andan ISVD that is made part of a polypeptide is said to be “formatted” orto be “in the format of” said polypeptide. Examples of ways in which anISVD may be formatted and examples of such formats will be clear to theskilled person based on the disclosure herein; and such formattedimmunoglobulin single variable domain form a further aspect of theinvention.

Preferred CDRs are depicted in Table A-2.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binding ADAMTS5 essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   (i) CDR1 is chosen from the group consisting of SEQ ID NOs: 21,        35, 20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34;        and amino acid sequences that have 1, 2 or 3 amino acid        difference(s) with SEQ ID NOs: 21, 35, 20, 22, 25, 33, 33, 28,        24, 23, 26, 27, 29, 30, 31, 32 and 34;    -   (ii) CDR2 is chosen from the group consisting of SEQ ID NOs: 37,        53, 36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48,        49 and 52; and amino acid sequences that have 1, 2 or 3 amino        acid difference(s) with SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44,        45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and    -   (iii) CDR3 is chosen from the group consisting of SEQ ID NO: SEQ        ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60,        64, 65, 66, 67 and 70; and amino acid sequences that have 1, 2,        3 or 4 amino acid difference(s) with SEQ ID NOs: 55, 118, 71,        54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67 and        70.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binding ADAMTS5 essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   (i) CDR1 is chosen from the group consisting of        -   (a) SEQ ID NO: 22; and        -   (b) amino acid sequence that has 1, 2, 3, 4, 5 or 6 amino            acid difference(s) with SEQ ID NO: 22, wherein            -   at position 2 the S has been changed into R;            -   at position 3 the A has been changed into T;            -   at position 4 the V has been changed into F;            -   at position 6 the V has been changed into S;            -   at position 7 the N has been changed into Y; and/or            -   at position 10 the A has been changed into G;    -   (ii) CDR2 is SEQ ID NO: 36; and    -   (iii) CDR3 is SEQ ID NO: 54.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binding ADAMTS5 essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   (i) CDR1 is SEQ ID NO: 33;    -   (ii) CDR2 is chosen from the group consisting of        -   (c) SEQ ID NO: 50; and        -   (d) amino acid sequence that has 1, 2, or 3 amino acid            difference(s) with SEQ ID NO: 50, wherein            -   at position 8 the M has been changed into I;            -   at position 9 the P has been changed into T; and/or            -   at position 10 the Y has been changed into F; and    -   (iii) CDR3 is chosen from the group consisting of        -   (e) SEQ ID NO: 68; and        -   (f) amino acid sequence that has 1 or 2 amino acid            difference(s) with SEQ ID NO: 68, wherein            -   at position 5 the F has been changed into L; and/or            -   at position 11 the D has been changed into E.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binding ADAMTS5 essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   (i) CDR1 is SEQ ID NO: 28;    -   (ii) CDR2 is chosen from the group consisting of        -   (c) SEQ ID NO: 44; and        -   (d) amino acid sequence that has 1, 2, or 3 amino acid            difference(s) with SEQ ID NO: 44, wherein            -   at position 3 the S has been changed into T;            -   at position 4 the R has been changed into W;            -   at position 8 the T has been changed into I; and/or            -   at position 9 the T has been changed into L; and    -   (iii) CDR3 is chosen from the group consisting of        -   (e) SEQ ID NO: 62; and        -   (f) amino acid sequence that has 1 or 2 amino acid            difference(s) with SEQ ID NO: 62, wherein            -   at position 1 the G has been changed into S; and/or            -   at position 14 the D has been changed into E.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binds ADAMTS5 and essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich

-   -   CDR1 is chosen from the group consisting of SEQ ID NOs: 21, 35,        20, 22, 25, 33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34;    -   CDR2 is chosen from the group consisting of SEQ ID NOs: 37, 53,        36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49        and 52; and    -   CDR3 is chosen from the group consisting of SEQ ID NOs: 55, 118,        71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65, 66, 67        and 70.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binds ADAMTS5 and essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich said ISVD is chosen from the group of ISVDs, wherein:

-   -   CDR1 is SEQ ID NO: 21, CDR2 is SEQ ID NO: 37 and CDR3 is SEQ ID        NO: 55;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO: 53 and CDR3 is SEQ ID        NO: 118;    -   CDR1 is SEQ ID NO: 35, CDR2 is SEQ ID NO:53 and CDR3 is SEQ ID        NO:71;    -   CDR1 is SEQ ID NO: 20, CDR2 is SEQ ID NO: 36 and CDR3 is SEQ ID        NO: 54;    -   CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 36 and CDR3 is SEQ ID        NO: 54;    -   CDR1 is SEQ ID NO: 25, CDR2 is SEQ ID NO: 40 and CDR3 is SEQ ID        NO: 58;    -   CDR1 is SEQ ID NO:33, CDR2 is SEQ ID NO:50 and CDR3 is SEQ ID        NO:68;    -   CDR1 is SEQ ID NO: 33, CDR2 is SEQ ID NO: 51 and CDR3 is SEQ ID        NO: 69;    -   CDR1 is SEQ ID NO:28, CDR2 is SEQ ID NO:44 and CDR3 is SEQ ID        NO:62;    -   CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 45 and CDR3 is SEQ ID        NO: 63;    -   CDR1 is SEQ ID NO: 28, CDR2 is SEQ ID NO: 43 and CDR3 is SEQ ID        NO: 61;    -   CDR1 is SEQ ID NO: 24, CDR2 is SEQ ID NO: 39 and CDR3 is SEQ ID        NO: 57;    -   CDR1 is SEQ ID NO: 23, CDR2 is SEQ ID NO: 38 and CDR3 is SEQ ID        NO: 56;    -   CDR1 is SEQ ID NO:26, CDR2 is SEQ ID NO:41 and CDR3 is SEQ ID        NO:59;    -   CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 119 and CDR3 is SEQ ID        NO: 60;    -   CDR1 is SEQ ID NO: 27, CDR2 is SEQ ID NO: 42 and CDR3 is SEQ ID        NO: 60;    -   CDR1 is SEQ ID NO:29, CDR2 is SEQ ID NO:46 and CDR3 is SEQ ID        NO:64;    -   CDR1 is SEQ ID NO:30, CDR2 is SEQ ID NO:47 and CDR3 is SEQ ID        NO:65;    -   CDR1 is SEQ ID NO: 31, CDR2 is SEQ ID NO: 48 and CDR3 is SEQ ID        NO: 66;    -   CDR1 is SEQ ID NO: 32, CDR2 is SEQ ID NO: 49 and CDR3 is SEQ ID        NO: 67; and    -   CDR1 is SEQ ID NO:34, CDR2 is SEQ ID NO:52 and CDR3 is SEQ ID        NO:70.

In a particular preferred aspect, the present invention relates to anISVD as described herein, wherein said ISVD specifically binds ADAMTS5and essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 is or comprises SEQ ID NO: 21, CDR2 is SEQID NO: 37 and CDR3 is SEQ ID NO: 55.

In particular, the present invention relates to an ISVD as describedherein, wherein said ISVD specifically binds ADAMTS5 and essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3 respectively), inwhich said ISVD is chosen from the group consisting of SEQ ID NO:s 2,116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and18.

It will be appreciated that, without limitation, the immunoglobulinsingle variable domains of the present invention may be used as a“building block” for the preparation of a polypeptide, which mayoptionally contain one or more further immunoglobulin single variabledomains that can serve as a building block (i.e., against the same oranother epitope on ADAMTS5 and/or against one or more other antigens,proteins or targets than ADAMTS5).

The polypeptide of the invention (also indicated herein as “Nanobodyconstruct”) comprises at least one ISVD binding an ADAMTS, preferablyADAMTS5, such as two ISVDs binding ADAMTS5, and preferably also an ISVDbinding Albumin. In a polypeptide of the invention, the ISVDs may bedirectly linked or linked via a linker. Even more preferably, thepolypeptide of the invention comprises a C-terminal extension. As willbe detailed herein, the C-terminal extension essentiallyprevents/removes binding of pre-existing antibodies/factors in mostsamples of human subjects/patients. The C-terminal extension is presentC-terminally of the last amino acid residue (usually a serine residue)of the last (most C-terminally located) ISVD.

As further elaborated infra, the ISVDs may be derived from a V_(HH),V_(H) or a V_(L) domain, however, the ISVDs are chosen such that they donot form complementary pairs of V_(H) and V_(L) domains in thepolypeptides of the invention. The Nanobody, V_(HH), and humanizedV_(HH) are unusual in that they are derived from natural camelidantibodies which have no light chains, and indeed these domains areunable to associate with camelid light chains to form complementaryV_(HH) and V_(L) pairs. Thus, the polypeptides of the present inventiondo not comprise complementary ISVDs and/or form complementary ISVDpairs, such as, for instance, complementary V_(H)/V_(L) pairs.

Generally, polypeptides or constructs that comprise or essentiallyconsist of a single building block, single ISVD or single Nanobody willbe referred to herein as “monovalent” polypeptides and “monovalentconstructs”, respectively. Polypeptides or constructs that comprise twoor more building blocks (such as e.g., ISVDs) will also be referred toherein as “multivalent” polypeptides or constructs, and the buildingblocks/ISVDs present in such polypeptides or constructs will also bereferred to herein as being in a “multivalent format”. For example, a“bivalent” polypeptide may comprise two ISVDs, optionally linked via alinker sequence, whereas a “trivalent” polypeptide may comprise threeISVDs, optionally linked via two linker sequences; whereas a“tetravalent” polypeptide may comprise four ISVDs, optionally linked viathree linker sequences, etc.

In a multivalent polypeptide, the two or more ISVDs may be the same ordifferent, and may be directed against the same antigen or antigenicdeterminant (for example against the same part(s) or epitope(s) oragainst different parts or epitopes) or may alternatively be directedagainst different antigens or antigenic determinants; or any suitablecombination thereof. Polypeptides and constructs that contain at leasttwo building blocks (such as e.g., ISVDs) in which at least one buildingblock is directed against a first antigen (i.e., ADAMTS5) and at leastone building block is directed against a second antigen (i.e., differentfrom ADAMTS5) will also be referred to as “multispecific” polypeptidesand constructs, and the building blocks (such as e.g., ISVDs) present insuch polypeptides and constructs will also be referred to herein asbeing in a “multispecific format”. Thus, for example, a “bispecific”polypeptide of the invention is a polypeptide that comprises at leastone ISVD directed against a first antigen (i.e., ADAMTS5) and at leastone further ISVD directed against a second antigen (i.e., different fromADAMTS5), whereas a “trispecific” polypeptide of the invention is apolypeptide that comprises at least one ISVD directed against a firstantigen (i.e., ADAMTS5), at least one further ISVD directed against asecond antigen (i.e., different from ADAMTS5) and at least one furtherISVD directed against a third antigen (i.e., different from both ADAMTS5and the second antigen); etc.

In an aspect, the present invention relates to a polypeptide, comprisingat least 2 ISVDs, wherein at least 1 ISVD specifically binds ADAMTS,preferably ADAMTS5, more preferably chosen from the group consisting ofSEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12,13, 14, 15 and 18.

In an aspect, the present invention relates to a polypeptide comprisingat least 2 ISVDs, wherein said at least 2 ISVDs specifically bindADAMTS, preferably ADAMTS5, more preferably each ISVD of said 2 ISVDs ischosen independently from the group consisting of SEQ ID NO:s 2, 116,19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

“Multiparatopic” polypeptides and “multiparatopic” constructs, such ase.g., “biparatopic” polypeptides or constructs and “triparatopic”polypeptides or constructs, comprise or essentially consist of two ormore building blocks that each have a different paratope.

Accordingly, the ISVDs of the invention that bind ADAMTS5 can be inessentially isolated form (as defined herein), or they may form part ofa construct or polypeptide, which may comprise or essentially consist ofone or more ISVD(s) that bind ADAMTS5 and which may optionally furthercomprise one or more further amino acid sequences (all optionally linkedvia one or more suitable linkers). The present invention relates to apolypeptide or construct that comprises or essentially consists of atleast one ISVD according to the invention, such as one or more ISVDs ofthe invention (or suitable fragments thereof), binding ADAMTS5.

The one or more ISVDs of the invention can be used as a building blockin such a polypeptide or construct, so as to provide a monovalent,multivalent or multiparatopic polypeptide or construct of the invention,respectively, all as described herein. The present invention thus alsorelates to a polypeptide which is a monovalent construct comprising oressentially consisting of one monovalent polypeptide or ISVD of theinvention.

The present invention thus also relates to a polypeptide or constructwhich is a multivalent polypeptide or multivalent construct,respectively, such as e.g., a bivalent or trivalent polypeptide orconstruct comprising or essentially consisting of two or more ISVDs ofthe invention (for multivalent and multispecific polypeptides containingone or more VHH domains and their preparation, reference is also made toConrath et al. (i. Biol. Chem. 276: 7346-7350, 2001), as well as to forexample WO 96/34103, WO 99/23221 and WO 2010/115998).

In an aspect, in its simplest form, the multivalent polypeptide orconstruct of the invention is a bivalent polypeptide or construct of theinvention comprising a first ISVD, such as a Nanobody, directed againstADAMTS5, and an identical second ISVD, such as a Nanobody, directedagainst ADAMTS5, wherein said first and said second ISVDs, such asNanobodies, may optionally be linked via a linker sequence (as definedherein). In another form, a multivalent polypeptide or construct of theinvention may be a trivalent polypeptide or construct of the invention,comprising a first ISVD, such as Nanobody, directed against ADAMTS5, anidentical second ISVD, such as Nanobody, directed against ADAMTS5 and anidentical third ISVD, such as a Nanobody, directed against ADAMTS5, inwhich said first, second and third ISVDs, such as Nanobodies, mayoptionally be linked via one or more, and in particular two, linkersequences. In an aspect, the invention relates to a polypeptide orconstruct that comprises or essentially consists of at least two ISVDsaccording to the invention, such as 2, 3 or 4 ISVDs (or suitablefragments thereof), binding ADAMTS5. The two or more ISVDs mayoptionally be linked via one or more peptidic linkers.

In another aspect, the multivalent polypeptide or construct of theinvention may be a bispecific polypeptide or construct of the invention,comprising a first ISVD, such as a Nanobody, directed against ADAMTS5,and a second ISVD, such as a Nanobody, directed against a secondantigen, such as, for instance, Aggrecan, in which said first and secondISVDs, such as Nanobodies, may optionally be linked via a linkersequence (as defined herein); whereas a multivalent polypeptide orconstruct of the invention may also be a trispecific polypeptide orconstruct of the invention, comprising a first ISVD, such as a Nanobody,directed against ADAMTS5, a second ISVD, such as a Nanobody, directedagainst a second antigen, such as for instance Aggrecan, and a thirdISVD, such as a Nanobody, directed against a third antigen, in whichsaid first, second and third ISVDs, such as Nanobodies, may optionallybe linked via one or more, and in particular two, linker sequences.

The invention further relates to a multivalent polypeptide thatcomprises or (essentially) consists of at least one ISVD (or suitablefragments thereof) binding ADAMTS5, preferably human ADAMTS5, and oneadditional ISVD, such as an ISVD binding Aggrecan.

Particularly preferred bivalent, bispecific polypeptides or constructsin accordance with the invention are those shown in the Examplesdescribed herein and in Table A-1 (cf. SEQ ID NO:s 120-130 (i.e. SEQ IDNO: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 and 130), mostpreferably SEQ ID NO:s 129 and 130).

In a preferred aspect, the polypeptide or construct of the inventioncomprises or essentially consists of at least two ISVDs, wherein said atleast two ISVDs can be the same or different, but of which at least oneISVD is directed against ADAMTS5, preferably said ISVD binding ADAMTS5is chosen from the group consisting of SEQ ID NO:s 2, 116, 19, 1, 3, 6,16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

The two or more ISVDs present in the multivalent polypeptide orconstruct of the invention may consist of a light chain variable domainsequence (e.g., a V_(L)-sequence) or of a heavy chain variable domainsequence (e.g., a V_(H)-sequence); they may consist of a heavy chainvariable domain sequence that is derived from a conventional four-chainantibody or of a heavy chain variable domain sequence that is derivedfrom heavy chain antibody. In a preferred aspect, they consist of aDomain antibody (or an amino acid that is suitable for use as a domainantibody), of a single domain antibody (or an amino acid that issuitable for use as a single domain antibody), of a “dAb” (or an aminoacid that is suitable for use as a dAb), of a Nanobody® (including butnot limited to V_(HH)), of a humanized V_(HH) sequence, of a camelizedV_(H) sequence; or of a V_(HH) sequence that has been obtained byaffinity maturation. The two or more immunoglobulin single variabledomains may consist of a partially or fully humanized Nanobody or apartially or fully humanized VHH.

In an aspect of the invention, the first ISVD and the second ISVDpresent in the multiparatopic (preferably biparatopic or triparatopic)polypeptide or construct of the invention do not (cross)-compete witheach other for binding to ADAMTS5 and, as such, belong to differentfamilies. Accordingly, the present invention relates to a multiparatopic(preferably biparatopic) polypeptide or construct comprising two or moreISVDs wherein each ISVD belongs to a different family. In an aspect, thefirst ISVD of this multiparatopic (preferably biparatopic) polypeptideor construct of the invention does not cross-block the binding toADAMTS5 of the second ISVD of this multiparatopic (preferablybiparatopic) polypeptide or construct of the invention and/or the firstISVD is not cross-blocked from binding to ADAMTS5 by the second ISVD. Inanother aspect, the first ISVD of a multiparatopic (preferablybiparatopic) polypeptide or construct of the invention cross-blocks thebinding to ADAMTS5 of the so second ISVD of this multiparatopic(preferably biparatopic) polypeptide or construct of the inventionand/or the first ISVD is cross-blocked from binding to ADAMTS5 by thesecond ISVD.

In a particularly preferred aspect, the polypeptide or construct of theinvention comprises or essentially consists of three or more ISVDs, ofwhich at least two ISVDs are directed against ADAMTS5. It will beappreciated that said at least two ISVDs directed against ADAMTS5 can bethe same or different, can be directed against the same epitope ordifferent epitopes of ADAMTS5, can belong to the same epitope bin or todifferent epitope bins, and/or can bind to the same or different domainsof ADAMTS5.

The relative affinities may depend on the location of the ISVDs in thepolypeptide. It will be appreciated that the order of the ISVDs in apolypeptide of the invention (orientation) may be chosen according tothe needs of the person skilled in the art. The order of the individualISVDs as well as whether the polypeptide comprises a linker is a matterof design choice. Some orientations, with or without linkers, mayprovide preferred binding characteristics in comparison to otherorientations. For instance, the order of a first ISVD (e.g. ISVD 1) anda second ISVD (e.g. ISVD 2) in the polypeptide of the invention may be(from N-terminus to C-terminus): (i) ISVD 1 (e.g. Nanobody1)-[linker]-ISVD 2 (e.g. Nanobody 2)-[C-terminal extension]; or (ii)ISVD 2 (e.g. Nanobody 2)-[linker]-ISVD 1 (e.g. Nanobody 1)-[C-terminalextension]; (wherein the moieties between the square brackets, i.e.linker and C-terminal extension, are optional). All orientations areencompassed by the invention. Polypeptides that contain an orientationof ISVDs that provides desired binding characteristics may be easilyidentified by routine screening, for instance as exemplified in theexamples section. A preferred order is from N-terminus to C-terminus:ISVD binding ADAMTS5-[linker]-ISVD binding Albumin orAggrecan-[C-terminal extension], wherein the moieties between the squarebrackets are optional.

In an aspect, the present invention relates to a polypeptide comprisingtwo or more ISVDs which specifically bind ADAMTS5, wherein

-   a) at least a “first” ISVD specifically binds a first antigenic    determinant, epitope, part, domain, subunit or conformation of    ADAMTS5, preferably said “first” ISVD specifically binding ADAMTS5    is chosen from the group consisting of SEQ ID NO:s 2, 1, 3, 6, 16,    17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13, 14, 15 and 18; and-   b) at least a “second” ISVD specifically binds a second antigenic    determinant, epitope, part, domain, subunit or conformation of    ADAMTS5, different from the first antigenic determinant epitope,    part, domain, subunit or conformation, respectively, preferably said    “second” ISVD specifically binding ADAMTS5 is SEQ ID NO: 116 or 19.

In a preferred aspect, the polypeptide or construct of the inventioncomprises or essentially consists of at least two ISVDs binding ADAMTS5,wherein said at least two ISVDs can be the same or different, which areindependently chosen from the group consisting of SEQ ID NO:s 2, 116,19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18.

In a further aspect, the invention relates to a multiparatopic(preferably biparatopic) polypeptide or construct comprising two or moreISVDs directed against ADAMTS5 that bind the same epitope(s) as is boundby any one of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4,7, 117, 8, 12, 13, 14, 15 and 18.

In a further aspect, the invention relates to a polypeptide as describedherein, wherein said polypeptide has at least 80%, 90%, 95% or 100%sequence identity with any of SEQ ID NO:s 1-19 (i.e. SEQ ID NO: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19), 116-117 or120-130 (i.e. SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128,129 and 130).

In an aspect, the present invention relates to a polypeptide asdescribed herein, which is chosen from the group consisting of SEQ IDNO: 127 (clone 130 049-093-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb),SEQ ID NO: 127 (clone 130 049-093-Alb) and SEQ ID NO: 128 (clone1319D3-093-Alb).

The art is in need of more effective therapies for disorders affectingcartilage in joints, such as osteoarthritis. Especially whenadministered systematically, the residence time of most drugs isinsufficient. The present inventors hypothesized that the efficacy of atherapeutic drug, such as a construct, polypeptide and ISVD of theinvention, could be increased significantly by coupling the therapeuticdrug to a moiety which extends the half-life of the drug andconsequently increase retention of the drug, but which should notdisrupt the efficacy of said therapeutic drug.

In a specific aspect of the invention, a construct or polypeptide of theinvention may have a moiety conferring an increased half-life, comparedto the corresponding construct or polypeptide of the invention withoutsaid moiety. Some preferred, but non-limiting examples of suchconstructs and polypeptides of the invention will become clear to theskilled person based on the further disclosure herein, and for examplecomprise ISVDs or polypeptides of the invention that have beenchemically modified to increase the half-life thereof (for example, bymeans of pegylation); ADAMTS5 binders of the invention, such as ISVDsand/or polypeptides of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin); or polypeptides of the invention which comprise at least oneISVD of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) which increases thehalf-life of the amino acid sequence of the invention. Examples ofconstructs of the invention, such as polypeptides of the invention,which comprise such half-life extending moieties or ISVDs will becomeclear to the skilled person based on the further disclosure herein; andfor example include, without limitation, polypeptides in which the oneor more ISVDs of the invention are suitably linked to one or more serumproteins or fragments thereof (such as (human) serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, domain antibodies, immunoglobulinsingle variable domains that are suitable for use as a domain antibody,single domain antibodies, immunoglobulin single variable domains thatare suitable for use as a single domain antibody, dAbs, immunoglobulinsingle variable domains that are suitable for use as a dAb, orNanobodies that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrin; reference is made to the further description and referencesmentioned herein); polypeptides in which an amino acid sequence of theinvention is linked to an Fc portion (such as a human Fc) or a suitablepart or fragment thereof; or polypeptides in which the one or moreimmunoglobulin single variable domains of the invention are suitablelinked to one or more small proteins or peptides that can bind to serumproteins, such as, without limitation, the proteins and peptidesdescribed in WO 91/01743, WO 01/45746, WO 02/076489, WO2008/068280,WO2009/127691 and PCT/EP2011/051559.

In an aspect the present invention provides a construct of the inventionor a polypeptide, wherein said construct or said polypeptide furthercomprises a serum protein binding moiety or a serum protein. Preferably,said serum protein binding moiety binds serum albumin, such as humanserum albumin.

In an aspect, the present invention relates to a polypeptide asdescribed herein, comprising an ISVD binding serum albumin.

Generally, the constructs or polypeptides of the invention withincreased half-life preferably have a half-life that is at least 1.5times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, greater than thehalf-life of the corresponding constructs or polypeptides of theinvention per se, i.e. without the moiety conferring the increasedhalf-life. For example, the constructs or polypeptides of the inventionwith increased half-life may have a half-life e.g., in humans that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding constructs orpolypeptides of the invention per se, i.e. without the moiety conferringthe increased half-life.

In a preferred, but non-limiting aspect of the invention, the constructsof the invention and polypeptides of the invention, have a serumhalf-life e.g. in humans that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding constructs or polypeptides of the invention per se,i.e. without the moiety conferring the increased half-life.

In another preferred, but non-limiting aspect of the invention, suchconstructs of the invention, such as polypeptides of the invention,exhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, constructs orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days).

In a particularly preferred but non-limiting aspect of the invention,the invention provides a construct of the invention and a polypeptide ofthe invention, comprising besides the one or more building blocksbinding ADAMTS5 at least one building block binding serum albumin, suchas an ISVD binding serum albumin, such as human serum albumin asdescribed herein. Preferably, said ISVD binding serum albumin comprisesor essentially consists of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 is SFGMS, CDR2 is SISGSGSDTLYADSVKG andCDR3 is GGSLSR. Preferably, said ISVD binding human serum albumin ischosen from the group consisting of Alb8, Alb23, Alb129, Alb132, Alb11,Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG,Alb82-GGG, Alb92 or Alb223 (cf. Table D).

In an aspect, the present invention relates to a polypeptide asdescribed herein, comprising at least one ISVD binding ADAMTS5 and anISVD binding serum albumin, preferably chosen from the group consistingof SEQ ID NO: 129 (clone 577 2F3*-Alb), SEQ ID NO: 130 (clone 5792F3*-093-Alb), SEQ ID NO: 120 (clone 4 2A12-Alb), SEQ ID NO: 121 (clone5 2D7-Alb), SEQ ID NO: 122 (clone 6 2F3-Alb), SEQ ID NO: 123 (clone 69049-Alb), SEQ ID NO: 124 (clone 70 9D3-Alb), SEQ ID NO: 125 (clone 713B2-Alb), SEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone130 049-093-Alb), and SEQ ID NO: 128 (clone 1319D3-093-Alb)(cf. TableA-1).

In an embodiment, the present invention relates to construct of theinvention, such as a polypeptide comprising a serum protein bindingmoiety, wherein said serum protein binding moiety is a non-antibodybased polypeptide.

The art is in need of more effective therapies for disorders affectingcartilage in joints, such as osteoarthritis. Even when administeredintra-articularly, the residence time of most drugs for treatingaffected cartilage is insufficient. The present inventors hypothesizedthat the efficacy of a therapeutic drug, such as a construct,polypeptide and ISVD of the invention, may be modulated by coupling thetherapeutic drug to a moiety which would “anchor” the drug in the jointand consequently increase retention of the drug, but which should notdisrupt the efficacy of said therapeutic drug (this moiety is hereinalso indicated as “cartilage anchoring protein” or “CAP”). Thisanchoring concept could not only modulate the efficacy of a drug, butalso the operational specificity for a diseased joint by decreasingtoxicity and side-effects, thus widening the number of possible usefuldrugs.

It was anticipated that a format of a molecule for clinical usecomprises one or two building blocks, such as ISVDs, binding ADAMTS5 andone or more building blocks, e.g. ISVDs, with such a retention mode ofaction, and possibly further moieties. In the co-pending application itis demonstrated that such formats retain both ADAMTS5 binding and atherapeutic effect, e.g. inhibitory activity, as well as retentionproperties. The one or more building blocks, such as ISVDs, with aretention mode of action can be any building block having a retentioneffect (“CAP building block”) in diseases in which ADAMTS5 is involved,such as arthritic disease, osteoarthritis, spondyloepimetaphysealdysplasia, lumbar disk degeneration disease, Degenerative joint disease,rheumatoid arthritis, osteochondritis dissecans, aggrecanopathies.

A “CAP building block” is used for directing, anchoring and/or retainingother, e.g. therapeutic, building blocks, such as ISVDs binding ADAMTS5at a desired site, such as e.g. in a joint, in which said other, e.g.therapeutic, building block is to exert its effect, e.g. binding and/orinhibiting ADAMTS5.

The present inventors further hypothesized that Aggrecan binders, suchas ISVD(s) binding Aggrecan might potentially function as such ananchor, although Aggrecan is heavily glycosylated and degraded invarious disorders affecting cartilage in joints. Moreover, in view ofthe costs and extensive testing in various animal models required beforea drug can enter the clinic, such Aggrecan binders should preferentiallyhave a broad cross-reactivity, e.g. the Aggrecan binders should bind toAggrecan of various species.

Using various ingenious immunization, screening and characterizationmethods, the present inventors were able to identify various Aggrecanbinders with superior selectivity, stability and specificity features,which enabled prolonged retention and activity in the joint (cf.co-pending application).

In an aspect, the present invention relates to a method for reducingand/or inhibiting the efflux of a composition, a polypeptide or aconstruct from a joint, wherein said method comprises administering apharmaceutically active amount of at least one polypeptide according tothe invention, a construct according to the invention, or a compositionaccording to the invention to a person in need thereof.

In the present invention the term “reducing and/or inhibiting theefflux” means reducing and/or inhibiting the outward flow of thecomposition, polypeptide or construct from within a joint to theoutside. Preferably, the efflux is reduced and/or inhibited by at least10% such as at least 20%, 30%, 40% or 50% or even more such as at least60%, 70%, 80%, 90% or even 100%, compared to the efflux of theaforementioned composition, polypeptide or construct in a joint underthe same conditions but without the presence of the Aggrecan binder ofthe invention, e.g. ISVD(s) binding Aggrecan.

Next to the diseases in which ADAMTS5 is involved, such as arthriticdisease, osteoarthritis, spondyloepimetaphyseal dysplasia, lumbar diskdegeneration disease, Degenerative joint disease, rheumatoid arthritis,osteochondritis dissecans and aggrecanopathies it is anticipated thatthe Aggrecan binders of the invention can also be used in various otherdiseases affecting cartilage, such as arthropathies andchondrodystrophies, arthritic disease (such as osteoarthritis,rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumaticrupture or detachment), achondroplasia, costochondritis,Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar diskdegeneration disease, degenerative joint disease, and relapsingpolychondritis (commonly indicated herein as “Aggrecan associateddiseases”).

Said CAP building block, e.g. ISVD(s) binding Aggrecan, preferably bindsto cartilaginous tissue such as cartilage and/or meniscus. In apreferred aspect, the CAP building block is cross-reactive for otherspecies and specifically binds one or more of human Aggrecan (SEQ ID NO:155), dog Aggrecan, bovine Aggrecan, rat Aggrecan; pig Aggrecan; mouseAggrecan, rabbit Aggrecan; cynomolgus Aggrecan and/or rhesus Aggrecan.Relevant structural information for Aggrecan may be found, for example,at (UniProt) Accession Numbers as depicted in the Table 2 below.

A preferred CAP building block is an ISVD binding Aggrecan, preferablyhuman Aggrecan, preferably represented by SEQ ID NO: 155 as depicted inTable B.

TABLE 2 name accession number human Aggrecan (SEQ ID NO: 155) P16112 dogAggrecan Q28343 bovine Aggrecan P13608 rat Aggrecan P07897 pig Aggrecan(core) Q29011 mouse Aggrecan Q61282 rabbit Aggrecan G1U677-1 cynomolgusAggrecan XP_002804990.1 rhesus Aggrecan XP_002804990.1

The present invention thus pertains to a polypeptide or constructaccording to the invention, further comprising at least one CAP buildingblock.

The present invention thus pertains to a polypeptide or constructaccording to the invention, further comprising at least one ISVDspecifically binding Aggrecan, preferably chosen from the ISVDsrepresented by SEQ ID NO:s 156 and 157.

In an aspect the present invention relates to a polypeptide as describedherein, comprising at least 2 ISVDs specifically binding Aggrecan.

In an aspect the present invention relates to a polypeptide as describedherein, comprising at least 2 ISVDs specifically binding Aggrecan,wherein said at least 2 ISVDs specifically binding Aggrecan can be thesame or different.

In an aspect the present invention relates to a polypeptide as describedherein, comprising at least 2 ISVDs specifically binding Aggrecan,wherein said at least 2 ISVDs specifically binding Aggrecan areindependently chosen from the group consisting of SEQ ID NOs: 156-157.

In an aspect the present invention relates to a polypeptide as describedherein, comprising at least 2 ISVDs specifically binding Aggrecan,wherein said at least 2 ISVDs specifically binding Aggrecan arerepresented by SEQ ID NO:s 156-157.

In an aspect the present invention relates to a polypeptide as describedherein, comprising an ISVD specifically binding Aggrecan, wherein saidISVD specifically binding Aggrecan, specifically binds to human Aggrecan[SEQ ID NO: 155].

In an aspect the present invention relates to a polypeptide as describedherein, wherein said ISVD specifically binding Aggrecan, specificallybinds human Aggrecan (SEQ ID NO: 155), dog Aggrecan, bovine Aggrecan,rat Aggrecan; pig Aggrecan; mouse Aggrecan, rabbit Aggrecan; cynomolgusAggrecan and/or rhesus Aggrecan.

In an aspect the present invention relates to a polypeptide as describedherein, wherein said ISVD specifically binding Aggrecan preferably bindsto cartilaginous tissue such as cartilage and/or meniscus.

It will be appreciated that the ISVD, polypeptide and construct of theinvention is preferably stable. The stability of a polypeptide,construct or ISVD of the invention can be measured by routine assaysknown to the person skilled in the art. Typical assays include (withoutbeing limiting) assays in which the activity of said polypeptide,construct or ISVD is determined, followed by incubating in SynovialFluid for a desired period of time, after which the activity isdetermined again.

In an aspect the present invention relates to an ISVD, polypeptide orconstruct of the invention having a stability of at least 7 days, suchas at least 14 days, 21 days, 1 month, 2 months or even 3 months insynovial fluid (SF) at 37° C.

The desired activity of the therapeutic building block, e.g. ISVDbinding ADAMTS5 in the multivalent polypeptide or construct of theinvention can be measured by routine assays known to the person skilledin the art.

In an aspect, the present invention relates to a construct as describedherein comprising at least one ISVD or polypeptide and one or more othergroups, residues, moieties or binding units. The one or more othergroups, residues, moieties or binding units are preferably chosen fromthe group consisting of a polyethylene glycol molecule, serum proteinsor fragments thereof, binding units that can bind to serum proteins, anFc portion, and small proteins or peptides that can bind to serumproteins, further amino acid residues, tags or other functionalmoieties, e.g., toxins, labels, radiochemicals, etc.

In an embodiment, as mentioned infra, the present invention relates to aconstruct of the invention, such as a polypeptide comprising a moietyconferring half-life extension, wherein said moiety is a PEG. Hence, thepresent invention relates also to a construct or polypeptide of theinvention comprising PEG.

The further amino acid residues may or may not change, alter orotherwise influence other (biological) properties of the polypeptide ofthe invention and may or may not add further functionality to thepolypeptide of the invention. For example, such amino acid residues:

-   a) can comprise an N-terminal Met residue, for example as result of    expression in a heterologous host cell or host organism.-   b) may form a signal sequence or leader sequence that directs    secretion of the polypeptide from a host cell upon synthesis (for    example to provide a pre-, pro- or prepro-form of the polypeptide of    the invention, depending on the host cell used to express the    polypeptide of the invention). Suitable secretory leader peptides    will be clear to the skilled person, and may be as further described    herein. Usually, such a leader sequence will be linked to the    N-terminus of the polypeptide, although the invention in its    broadest sense is not limited thereto;-   c) may form a “tag”, for example an amino acid sequence or residue    that allows or facilitates the purification of the polypeptide, for    example using affinity techniques directed against said sequence or    residue. Thereafter, said sequence or residue may be removed (e.g.    by chemical or enzyatical cleavage) to provide the polypeptide (for    this purpose, the tag may optionally be linked to the amino acid    sequence or polypeptide sequence via a cleavable linker sequence or    contain a cleavable motif). Some preferred, but non-limiting    examples of such residues are multiple histidine residues,    glutathione residues and a myc-tag such as AAAEQKLISEEDLNGAA;-   d) may be one or more amino acid residues that have been    functionalized and/or that can serve as a site for attachment of    functional groups. Suitable amino acid residues and functional    groups will be clear to the skilled person and include, but are not    limited to, the amino acid residues and functional groups mentioned    herein for the derivatives of the polypeptides of the invention.

Also encompassed in the present invention are constructs comprising apolypeptide and/or ISVD of the invention, which further comprise otherfunctional moieties, e.g., toxins, labels, radiochemicals, etc.

The other groups, residues, moieties or binding units may for example bechemical groups, residues, moieties, which may or may not by themselvesbe biologically and/or pharmacologically active. For example, andwithout limitation, such groups may be linked to the one or more ISVDsor polypeptides of the invention so as to provide a “derivative” of thepolypeptide or construct of the invention.

Accordingly, the invention in its broadest sense also comprisesconstructs and/or polypeptides that are derivatives of the constructsand/or polypeptides of the invention. Such derivatives can generally beobtained by modification, and in particular by chemical and/orbiological (e.g., enzymatic) modification, of the constructs and/orpolypeptides of the invention and/or of one or more of the amino acidresidues that form a polypeptide of the invention.

Examples of such modifications, as well as examples of amino acidresidues within the polypeptide sequences that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person (see also Zangi etal., Nat Biotechnol 31(10):898-907, 2013).

For example, such a modification may involve the introduction (e.g., bycovalent linking or in any other suitable manner) of one or more(functional) groups, residues or moieties into or onto the polypeptideof the invention, and in particular of one or more functional groups,residues or moieties that confer one or more desired properties orfunctionalities to the construct and/or polypeptide of the invention.Examples of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g., bycovalent binding or in any other suitable manner) of one or morefunctional moieties that increase the half-life, the solubility and/orthe absorption of the construct or polypeptide of the invention, thatreduce the immunogenicity and/or the toxicity of the construct orpolypeptide of the invention, that eliminate or attenuate anyundesirable side effects of the construct or polypeptide of theinvention, and/or that confer other advantageous properties to and/orreduce the undesired properties of the construct or polypeptide of theinvention; or any combination of two or more of the foregoing. Examplesof such functional moieties and of techniques for introducing them willbe clear to the skilled person, and can generally comprise allfunctional moieties and techniques mentioned in the general backgroundart cited hereinabove as well as the functional moieties and techniquesknown per se for the modification of pharmaceutical proteins, and inparticular for the modification of antibodies or antibody fragments(including ScFv's and single domain antibodies), for which reference isfor example made to Remington (Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa., 1980). Such functional moieties may forexample be linked directly (for example covalently) to a polypeptide ofthe invention, or optionally via a suitable linker or spacer, as willagain be clear to the skilled person.

One specific example is a derivative polypeptide or construct of theinvention wherein the polypeptide or construct of the invention has beenchemically modified to increase the half-life thereof (for example, bymeans of pegylation). This is one of the most widely used techniques forincreasing the half-life and/or reducing the immunogenicity ofpharmaceutical proteins and comprises attachment of a suitablepharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG)or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG).Generally, any suitable form of pegylation can be used, such as thepegylation used in the art for antibodies and antibody fragments(including but not limited to (single) domain antibodies and ScFv's);reference is made to, for example, Chapman (Nat. Biotechnol. 54:531-545, 2002), Veronese and Harris (Adv. Drug Deliv. Rev. 54: 453-456,2003), Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003) andWO 04/060965. Various reagents for pegylation of proteins are alsocommercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al. (Protein Engineering 16:761-770, 2003). For example, for this purpose, PEG may be attached to acysteine residue that naturally occurs in a polypeptide of theinvention, a construct or polypeptide of the invention may be modifiedso as to suitably introduce one or more cysteine residues for attachmentof PEG, or an amino acid sequence comprising one or more cysteineresidues for attachment of PEG may be fused to the N- and/or C-terminusof a construct or polypeptide of the invention, all using techniques ofprotein engineering known per se to the skilled person.

Preferably, for the constructs or polypeptides of the invention, a PEGis used with a molecular weight of more than 5000, such as more than10,000 and less than 200,000, such as less than 100,000; for example inthe range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the polypeptide or construct of theinvention. Suitable labels and techniques for attaching, using anddetecting them will be clear to the skilled person, and for exampleinclude, but are not limited to, fluorescent labels (such asfluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescentmetals such as ¹⁵²Eu or others metals from the lanthanide series),phosphorescent labels, chemiluminescent labels or bioluminescent labels(such as luminal, isoluminol, theromatic acridinium ester, imidazole,acridinium salts, oxalate ester, dioxetane or GFP and its analogs),radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co,⁵⁹Fe, and ⁷⁵Se), metals, metals chelates or metallic cations (forexample metallic cations such as ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru,⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metallic cations that areparticularly suited for use in in vivo, in vitro or in so situ diagnosisand imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe)), as well aschromophores and enzymes (such as malate dehydrogenase, staphylococcalnuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholine esterase). Other suitable labels will be clear to theskilled person, and for example include moieties that can be detectedusing NMR or ESR spectroscopy.

Such labelled polypeptides and constructs of the invention may, forexample, be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylene-diaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalmoiety that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional moiety may be usedto link the polypeptide of the invention to another protein, polypeptideor chemical compound that is bound to the other half of the bindingpair, i.e. through formation of the binding pair. For example, aconstruct or polypeptide of the invention may be conjugated to biotin,and linked to another protein, polypeptide, compound or carrierconjugated to avidin or streptavidin. For example, such a conjugatedconstruct or polypeptide of the invention may be used as a reporter, forexample in a diagnostic system where a detectable signal-producing agentis conjugated to avidin or streptavidin. Such binding pairs may forexample also be used to bind the construct or polypeptide of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example is the liposomal formulationsdescribed by Cao and Suresh (Journal of Drug Targeting 8: 257, 2000).Such binding pairs may also be used to link a therapeutically activeagent to the polypeptide of the invention.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw (Biotechnol.Appl. Biochem. 26: 143-151, 1997).

Preferably, the constructs, polypeptides and/or derivatives are suchthat they bind to ADAMTS5, with an affinity (suitably measured and/orexpressed as a K-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate or on-rate and/or k_(off) or off-rate, oralternatively as an IC₅₀ value, as further described herein) that is asdefined herein (e.g. as defined for the polypeptides of the invention).

Such constructs and/or polypeptides of the invention and derivativesthereof may also be in essentially isolated form (as defined herein).

In an aspect, the present invention relates to a construct of theinvention, that comprises or essentially consists of an ISVD accordingto the invention or a polypeptide according to the invention, and whichfurther comprises one or more other groups, residues, moieties orbinding units, which are optionally linked via one or more peptidiclinkers.

In an aspect, the present invention relates to a construct of theinvention, in which one or more other groups, residues, moieties orbinding units are chosen from the group consisting of a polyethyleneglycol molecule, serum proteins or fragments thereof, binding units thatcan bind to serum proteins, an Fc portion, and small proteins orpeptides that can bind to serum proteins.

In the constructs of the invention, such as the polypeptides of theinvention, the two or more building blocks, such as e.g. ISVDs, and theoptionally one or more other groups, drugs, agents, residues, moietiesor binding units may be directly linked to each other (as for exampledescribed in WO 99/23221) and/or may be linked to each other via one ormore suitable spacers or linkers, or any combination thereof. Suitablespacers or linkers for use in multivalent and multispecific polypeptideswill be clear to the skilled person, and may generally be any linker orspacer used in the art to link amino acid sequences. Preferably, saidlinker or spacer is suitable for use in constructing constructs,proteins or polypeptides that are intended for pharmaceutical use.

For instance, the polypeptide of the invention may, for example, be atrivalent, trispecific polypeptide, comprising one building block, suchas an ISVD binding ADAMTS5, an ISVD binding albumin, and potentiallyanother building block, such as a third ISVD, in which said first,second and third building blocks, such as ISVDs, may optionally belinked via one or more, and in particular 2, linker sequences. Also, thepresent invention provides a construct or polypeptide of the inventioncomprising a first ISVD binding ADAMTS5 and possibly a second ISVDbinding albumin and/or possibly a third ISVD and/or possibly a fourthISVD, wherein said first ISVD and/or said second ISVD and/or possiblysaid third ISVD and/or possibly said fourth ISVD are linked via linkers,in particular 3 linkers.

Some particularly preferred linkers include the linkers that are used inthe art to link antibody fragments or antibody domains. These includethe linkers mentioned in the general background art cited above, as wellas for example linkers that are used in the art to construct diabodiesor ScFv fragments (in this respect, however, it should be noted that,whereas in diabodies and in ScFv fragments, the linker sequence usedshould have a length, a degree of flexibility and other properties thatallow the pertinent V_(H) and V_(L) domains to come together to form thecomplete antigen-binding site, there is no particular limitation on thelength or the flexibility of the linker used in the polypeptide of theinvention, since each ISVD, such as Nanobodies, by itself forms acomplete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))₂, such as (for example (gly₄ser₂)₃or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30, GS15, GS9 andGS7 linkers described in the applications by Ablynx mentioned herein(see for example WO 06/040153 and WO 06/122825), as well as hinge-likeregions, such as the hinge regions of naturally occurring heavy chainantibodies or similar sequences (such as described in WO 94/04678).Preferred linkers are depicted in Table C.

Some other particularly preferred linkers are poly-alanine (such as A),as well as the linkers GS30 (see also SEQ ID NO: 85 in WO 06/122825) andGS9 (see also SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the final theconstruct of the invention, such as the polypeptide of the invention,including but not limited to the affinity, specificity or avidity for achemokine, or for one or more of the other antigens. Based on thedisclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific construct of the invention, suchas the polypeptide of the invention, optionally after some limitedroutine experiments.

For example, in multivalent polypeptides of the invention that comprisebuilding blocks, ISVDs or Nanobodies directed against ADAMTS5 andanother target, the length and flexibility of the linker are preferablysuch that it allows each building block, such as an ISVD, of theinvention present in the polypeptide to bind to its cognate target, e.g.the antigenic determinant on each of the targets. Again, based on thedisclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific construct of the invention, suchas a polypeptide of the invention, optionally after some limited routineexperiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to theconstructs of the invention, such as the polypeptides of the invention,and/or provide one or more sites for the formation of derivatives and/orfor the attachment of functional groups (e.g. as described herein forthe derivatives of the ISVDs of the invention). For example, linkerscontaining one or more charged amino acid residues can provide improvedhydrophilic properties, whereas linkers that form or contain smallepitopes or tags can be used for the purposes of detection,identification and/or purification. Again, based on the disclosureherein, the skilled person will be able to determine the optimal linkersfor use in a specific polypeptide of the invention, optionally aftersome limited routine experiments.

Finally, when two or more linkers are used in the constructs such aspolypeptides of the invention, these linkers may be the same ordifferent. Again, based on the disclosure herein, the skilled personwill be able to determine the optimal linkers for use in a specificconstruct or polypeptide of the invention, optionally after some limitedroutine experiments.

Usually, for easy of expression and production, a construct of theinvention, such as a polypeptide of the invention, will be a linearpolypeptide. However, the invention in its broadest sense is not limitedthereto. For example, when a construct of the invention, such as apolypeptide of the invention, comprises three of more building blocks,ISVDs or Nanobodies, it is possible to link them by use of a linker withthree or more “arms”, which each “arm” being linked to a building block,ISVD or Nanobody, so as to provide a “star-shaped” construct. It is alsopossible, although usually less preferred, to use circular constructs.

Accordingly, the present invention relates to a construct of theinvention, such as a polypeptide of the invention, wherein said ISVDsare directly linked to each other or are linked via a linker.

Accordingly, the present invention relates to a construct of theinvention, such as a polypeptide of the invention, wherein a first ISVDand/or a second ISVD and/or possibly an ISVD binding serum albumin arelinked via a linker.

Accordingly, the present invention relates to a construct of theinvention, such as a polypeptide of the invention, wherein said linkeris chosen from the group consisting of linkers of 3A, 5GS, 7GS, 9GS,10GS, 15GS, 18GS, 20GS, 25GS, 30GS, 35GS, poly-A, 8GS, 40GS, G1 hinge,9GS-G1 hinge, llama upper long hinge region, and G3 hinge, such as e.g.presented in Table C (SEQ ID NO:s 158-174).

Accordingly, the present invention relates to a construct of theinvention, such as a polypeptide of the invention, wherein saidpolypeptide is chosen from the group consisting of SEQ ID NOs: 120-130.

The invention further relates to methods for preparing the constructs,polypeptides, ISVDs, nucleic acids, host cells, and compositionsdescribed herein.

The multivalent polypeptides of the invention can generally be preparedby a method which comprises at least the step of suitably linking theISVD and/or monovalent polypeptide of the invention to one or morefurther ISVDs, optionally via the one or more suitable linkers, so as toprovide the multivalent polypeptide of the invention. Polypeptides ofthe invention can also be prepared by a method which generally comprisesat least the steps of providing a nucleic acid that encodes apolypeptide of the invention, expressing said nucleic acid in a suitablemanner, and recovering the expressed polypeptide of the invention. Suchmethods can be performed in a manner known per se, which will be clearto the skilled person, for example on the basis of the methods andtechniques further described herein.

A method for preparing multivalent polypeptides of the invention maycomprise at least the steps of linking two or more ISVDs of theinvention and for example one or more linkers together in a suitablemanner. The ISVDs of the invention (and linkers) can be coupled by anymethod known in the art and as further described herein. Preferredtechniques include the linking of the nucleic acid sequences that encodethe ISVDs of the invention (and linkers) to prepare a genetic constructthat expresses the multivalent polypeptide. Techniques for linking aminoacids or nucleic acids will be clear to the skilled person, andreference is again made to the standard handbooks, such as Sambrook etal. and Ausubel et al., mentioned above, as well as the Examples below.

Accordingly, the present invention also relates to the use of an ISVD ofthe invention in preparing a multivalent polypeptide of the invention.The method for preparing a multivalent polypeptide will comprise thelinking of an ISVD of the invention to at least one further ISVD of theinvention, optionally via one or more linkers. The ISVD of the inventionis then used as a binding domain or building block in providing and/orpreparing the multivalent polypeptide comprising 2 (e.g., in a bivalentpolypeptide), 3 (e.g., in a trivalent polypeptide), 4 (e.g., in atetravalent) or more (e.g., in a multivalent polypeptide) buildingblocks. In this respect, the ISVD of the invention may be used as abinding domain or binding unit in providing and/or preparing amultivalent, such as bivalent, trivalent or tetravalent polypeptide ofthe invention comprising 2, 3, 4 or more building blocks.

Accordingly, the present invention also relates to the use of an ISVDpolypeptide of the invention (as described herein) in preparing amultivalent polypeptide. The method for the preparation of themultivalent polypeptide will comprise the linking of the ISVD of theinvention to at least one further ISVD of the invention, optionally viaone or more linkers.

The polypeptides and nucleic acids of the invention can be prepared in amanner known per se, as will be clear to the skilled person from thefurther description herein. For example, the polypeptides of theinvention can be prepared in any manner known per se for the preparationof antibodies and in particular for the preparation of antibodyfragments (including but not limited to (single) domain antibodies andScFv fragments). Some preferred, but non-limiting methods for preparingthe polypeptides and nucleic acids include the methods and techniquesdescribed herein.

The method for producing a polypeptide of the invention may comprise thefollowing steps: the expression, in a suitable host cell or hostorganism (also referred to herein as a “host of the invention”) or inanother suitable expression system of a nucleic acid that encodes saidpolypeptide of the invention (also referred to herein as a “nucleic acidof the invention”); optionally followed by isolating and/or purifyingthe polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of: cultivatingand/or maintaining a host of the invention under conditions that aresuch that said host of the invention expresses and/or produces at leastone polypeptide of the invention; optionally followed by isolatingand/or purifying the polypeptide of the invention thus obtained.

Accordingly, the present invention also relates to a nucleic acid ornucleotide sequence that encodes a polypeptide, ISVD or construct of theinvention (also referred to as “nucleic acid of the invention”).

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA. According to one embodiment of the invention, thenucleic acid of the invention is in essentially isolated from, asdefined herein. The nucleic acid of the invention may also be in theform of, be present in and/or be part of a vector, e.g. expressionvector, such as for example a plasmid, cosmid or YAC, which again may bein essentially isolated form. Accordingly, the present invention alsorelates to an expression vector comprising a nucleic acid or nucleotidesequence of the invention.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the polypeptides of theinvention given herein, and/or can be isolated from a suitable sonatural source. Also, as will be clear to the skilled person, to preparea nucleic acid of the invention, also several nucleotide sequences, suchas at least two nucleic acids encoding ISVDs of the invention and forexample nucleic acids encoding one or more linkers can be linkedtogether in a suitable manner. Techniques for generating the nucleicacids of the invention will be clear to the skilled person and may forinstance include, but are not limited to, automated DNA synthesis;site-directed mutagenesis; combining two or more naturally occurringand/or synthetic sequences (or two or more parts thereof), introductionof mutations that lead to the expression of a truncated expressionproduct; introduction of one or more restriction sites (e.g. to createcassettes and/or regions that may easily be digested and/or ligatedusing suitable restriction enzymes), and/or the introduction ofmutations by means of a PCR reaction using one or more “mismatched”primers. These and other techniques will be clear to the skilled person,and reference is again made to the standard handbooks, such as Sambrooket al. and Ausubel et al., mentioned above, as well as to the Examplesbelow.

In a preferred but non-limiting embodiment, a genetic construct of theinvention comprises

-   a) at least one nucleic acid of the invention;-   b) operably connected to one or more regulatory elements, such as a    promoter and optionally a suitable terminator; and optionally also-   c) one or more further elements of genetic constructs known per se;    in which the terms “regulatory element”, “promoter”, “terminator”    and “operably connected” have their usual meaning in the art.

The genetic constructs of the invention may generally be provided bysuitably linking the nucleotide sequence(s) of the invention to the oneor more further elements described above, for example using thetechniques described in the general handbooks such as Sambrook et al.and Ausubel et al., mentioned above.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.,for expression and/or production of the polypeptide of the invention.Suitable hosts or host cells will be clear to the skilled person, andmay for example be any suitable fungal, prokaryotic or eukaryotic cellor cell line or any suitable fungal, prokaryotic or (non-human)eukaryotic organism as well as all other host cells or (non-human) hostsknown per se for the expression and production of antibodies andantibody fragments (including but not limited to (single) domainantibodies and ScFv fragments), which will be clear to the skilledperson. Reference is also made so to the general background art citedhereinabove, as well as to for example WO 94/29457; WO 96/34103; WO99/42077; Frenken et al. (Res Immunol. 149: 589-99, 1998); Riechmann andMuyldermans (1999), supra; van der Linden (J. Biotechnol. 80: 261-70,2000); Joosten et al. (Microb. Cell Fact. 2: 1, 2003); Joosten et al.(Appl. Microbiol. Biotechnol. 66: 384-92, 2005); and the furtherreferences cited herein. Furthermore, the polypeptides of the inventioncan also be expressed and/or produced in cell-free expression systems,and suitable examples of such systems will be clear to the skilledperson. Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above. The transformed host cell (which may be in the form ora stable cell line) or host organisms (which may be in the form of astable mutant line or strain) form further aspects of the presentinvention. Accordingly, the present invention relates to a host or hostcell comprising a nucleic acid according to the invention, or anexpression vector according to the invention. Preferably, these hostcells or host organisms are such that they express, or are (at least)capable of expressing (e.g., under suitable conditions), a polypeptideof the invention (and in case of a host organism: in at least one cell,part, tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the polypeptides of the invention, thetransformed host cell or transformed host organism may generally bekept, maintained and/or cultured under conditions such that the(desired) polypeptide of the invention is expressed/produced. Suitableconditions will be clear to the skilled person and will usually dependupon the host cell/host organism used, as well as on the regulatoryelements that control the expression of the (relevant) nucleotidesequence of the invention. Again, reference is made to the handbooks andpatent applications mentioned above in the paragraphs on the geneticconstructs of the invention.

The polypeptide of the invention may then be isolated from the hostcell/host organism and/or from the medium in which said host cell orhost organism was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g., using a specific,cleavable amino acid sequence fused with the polypeptide of theinvention) and/or preparative immunological techniques (i.e. usingantibodies against the polypeptide to be isolated).

In an aspect the invention relates to method for producing a construct,polypeptide or ISVD according to the invention comprising at least thesteps of: (a) expressing, in a suitable host cell or host organism or inanother suitable expression system, a nucleic acid sequence according tothe invention; optionally followed by (b) isolating and/or purifying theconstruct, polypeptide ISVD according to the invention.

In an aspect the invention relates to a composition comprising aconstruct, polypeptide, ISVD or nucleic acid according to the invention.

As mentioned supra, there remains a need for safe and efficacious OAmedicaments. Based on unconventional screening, characterization andcombinatory strategies, the present inventors identified ISVDs bindingand inhibiting ADAMTS5. These ADAMTS5 binders performed exceptionallywell in in vitro and in vivo experiments. Moreover, the ISVDs of theinvention were also demonstrated to be significantly more efficaciousthan the prior art compounds. The present invention thus provides ISVDsand polypeptides antagonizing ADAMTSs, in particular ADAMTS5, withimproved prophylactic, therapeutic and/or pharmacological properties,including a safer profile, compared to the prior art amino acidsequences and antibodies. In addition, these ADAMTS5 binders when linkedto ISVDs binding albumin had an increased retention in a subject, couldbe administered systematically while retaining activity.

In an aspect the present invention relates to a method of treating orprevention of diseases or disorders in an individual, for instance inwhich ADAMTS5 activity is involved, the method comprising administeringan ISVD or polypeptide according to the invention to said individual inan amount effective to treat or prevent a symptom of said disease ordisorder.

In an aspect the present invention relates to a composition according tothe invention, an ISVD according to the invention, a polypeptideaccording to the invention, and/or a construct according to theinvention for use as a medicament.

In another aspect, the invention relates to the use of an ISVD,polypeptide and/or construct of the invention in the preparation of apharmaceutical composition for prevention and/or treatment of at leastan ADAMTS5 associated disease; and/or for use in one or more of themethods of treatment mentioned herein.

The invention also relates to the use of an ISVD, polypeptide and/orconstruct of the invention in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of at least one diseaseor disorder that can be prevented and/or treated by modulating theactivity of an ADAMTS, preferably ADAMTS5 e.g. inhibiting Aggrecandegradation.

The invention also relates to the use of an ISVD, polypeptide, compoundand/or construct of the invention in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of at least one disease,disorder or condition that can be prevented and/or treated byadministering an ISVD, polypeptide, compound and/or construct of theinvention to a patient.

The invention further relates to an ISVD, polypeptide, compound and/orconstruct of the invention or a pharmaceutical composition comprisingthe same for use in the prevention and/or treatment of at least oneADAMTS5 associated disease.

It is anticipated that the ADAMTS5 binders of the invention can be usedin various diseases affecting cartilage, such as arthropathies andchondrodystrophies, arthritic disease, such as osteoarthritis,rheumatoid arthritis, gouty arthritis, psoriatic arthritis, traumaticrupture or detachment, achondroplasia, costochondritis,Spondyloepimetaphyseal dysplasia, spinal disc herniation, lumbar diskdegeneration disease, degenerative joint disease, and relapsingpolychondritis, osteochondritis dissecans and aggrecanopathies andnon-alcoholic steatohepatitis (NASH) (commonly indicated herein as“ADAMTS5 associated diseases”)

In an aspect the present invention relates to a composition, an ISVD, apolypeptide and/or a construct according to the invention for use intreating or preventing a symptom of an ADAMTS5 associated disease, suchas e.g. arthropathies and chondrodystrophies, arthritic disease, such asosteoarthritis, rheumatoid arthritis, gouty arthritis, psoriaticarthritis, traumatic rupture or detachment, achondroplasia,costochondritis, Spondyloepimetaphyseal dysplasia, spinal discherniation, lumbar disk degeneration disease, degenerative jointdisease, and relapsing polychondritis, osteochondritis dissecans andaggrecanopathies and NASH.

In an aspect the present invention relates to a method for preventing ortreating arthropathies and chondrodystrophies, arthritic disease, suchas osteoarthritis, rheumatoid arthritis, gouty arthritis, psoriaticarthritis, traumatic rupture or detachment, achondroplasia,costo-chondritis, Spondyloepimetaphyseal dysplasia, spinal discherniation, lumbar disk degeneration disease, degenerative jointdisease, and relapsing polychondritis and NASH wherein said methodcomprises administering, to a subject in need thereof, apharmaceutically active amount of at least a composition,immunoglobulin, polypeptide, or construct according to the invention toa person in need thereof.

In an aspect the present invention relates to the use of an ISVD,polypeptide, composition or construct according to the invention, in thepreparation of a pharmaceutical composition for treating or preventingsuch as arthropathies and chondrodystrophies, arthritic disease, such asosteoarthritis, rheumatoid arthritis, gouty arthritis, psoriaticarthritis, traumatic rupture or detachment, achondroplasia,costochondritis, Spondyloepimetaphyseal dysplasia, spinal discherniation, lumbar disk degeneration disease, degenerative jointdisease, and relapsing polychondritis, osteochondritis dissecans andaggrecanopathies and NASH.

It is also expected that by binding to Aggrecan, the constructs and/orpolypeptides of the invention may reduce or inhibit an activity of amember of the serine protease family, cathepsins, matrixmetallo-proteinases (MMPs), such as e.g. MMP20, but also ADAMTS4(Aggrecanase-1) and/or ADAMTS11 in degrading Aggrecan.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosage for anyone patient depends upon many factors, including the patient's size,weight, body surface area, age, the particular compound to beadministered, the activity of the employed polypeptide (includingantibodies), time and route of administration, general health, andcombination with other therapies or treatments. Proteinaceouspharmaceutically active matter may be present in amounts between 1 g and100 mg/kg body weight per dose; however, doses below or above thisexemplary range are also envisioned. If the regimen is a continuousinfusion, it may be in the range of 1 pg to 100 mg per kilogram of bodyweight per minute.

An ISVD, polypeptide or construct of the invention may be employed at aconcentration of, e.g., 0.01, 0.1, 0.5, 1, 2, 5, 10, 20 or 50 pg/ml inorder to inhibit and/or neutralize a biological function of ADAMTS5 byat least about 50%, preferably 75%, more preferably 90%, 95% or up to99%, and most preferably approximately 100% (essentially completely) asassayed by methods well known in the art.

Generally, the treatment regimen will comprise the administration of oneor more ISVDs, polypeptides and/or constructs of the invention, or ofone or more compositions comprising the same, in one or morepharmaceutically effective amounts or doses. The specific amount(s) ordoses to be administered can be determined by the clinician, again basedon the factors cited above. Useful dosages of the constructs,polypeptides, and/or ISVDs of the invention can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Generally, depending on the specific disease, disorder or condition tobe treated, the potency of the specific ISVD, polypeptide and/orconstruct of the invention to be used, the specific route ofadministration and the specific pharmaceutical formulation orcomposition used, the clinician will be able to determine a suitabledosing regimen.

The amount of the constructs, polypeptides, and/or ISVDs of theinvention required for use in treatment will vary not only with theparticular immunoglobulin, polypeptide, compound and/or constructselected but also with the route of administration, the nature of thecondition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician. Also the dosage of the constructs, polypeptides, and/or ISVDsof the invention varies depending on the target cell, tumor, tissue,graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

Usually, in the above method, an ISVD, polypeptide and/or construct ofthe invention will be used. It is however within the scope of theinvention to use two or more ISVDs, polypeptides and/or constructs ofthe invention in combination.

The ISVDs, polypeptides and/or constructs of the invention may be usedin combination with one or more further pharmaceutically activecompounds or principles, i.e., as a combined treatment regimen, whichmay or may not lead to a synergistic effect.

The pharmaceutical composition may also comprise at least one furtheractive agent, e.g. one or more further antibodies or antigen-bindingfragments thereof, peptides, proteins, nucleic acids, organic andinorganic molecules.

Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgment.

In particular, the ISVDs, polypeptides and/or constructs of theinvention may be used in combination with other pharmaceutically activecompounds or principles that are or can be used for the preventionand/or treatment of the diseases, disorders and conditions cited herein,as a result of which a synergistic effect may or may not be obtained.Examples of such compounds and principles, as well as routes, methodsand pharmaceutical formulations or compositions for administering themwill be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease, disorder or condition involved, as will be clear to theclinician. The clinician will also be able, where appropriate and on acase-by-case basis, to change or modify a particular treatment regimen,so as to achieve the desired therapeutic effect, to avoid, limit orreduce unwanted side-effects, and/or to achieve an appropriate balancebetween achieving the desired therapeutic effect on the one hand andavoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one construct of the invention, atleast one polypeptide of the invention, at least one ISVD of theinvention, or at least one nucleic acid of the invention and at leastone suitable carrier, diluent or excipient (i.e., suitable forpharmaceutical use), and optionally one or more further activesubstances. In a particular aspect, the invention relates to apharmaceutical composition that comprises a construct, polypeptide, ISVDor nucleic acid according to the invention, preferably at least one ofSEQ ID NOs: OOO and at least one suitable carrier, diluent or excipient(i.e., suitable for pharmaceutical use), and optionally one or morefurther active substances.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. In veterinaryapplications, the subject to be treated includes any animal raised forcommercial purposes or kept as a pet. As will be clear to the skilledperson, the subject to be treated will in particular be a personsuffering from, or at risk of, the diseases, disorders and conditionsmentioned herein. Hence, in a preferred embodiment of the invention, thepharmaceutical compositions comprising a polypeptide of the inventionare for use in medicine or diagnostics. Preferably, the pharmaceuticalcompositions are for use in human medicine, but they may also be usedfor veterinary purposes.

Again, in such a pharmaceutical composition, the one or moreimmunoglobulins, polypeptides, compounds and/or constructs of theinvention, or nucleotide encoding the same, and/or a pharmaceuticalcomposition comprising the same, may also be suitably combined with oneor more other active principles, such as those mentioned herein.

The invention also relates to a composition (such as, withoutlimitation, a pharmaceutical composition or preparation as furtherdescribed herein) for use, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multi-cellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers froma disease, disorder or condition of the invention).

It is to be understood that reference to treatment includes bothtreatment of established symptoms and prophylactic treatment, unlessexplicitly stated otherwise.

Generally, for pharmaceutical use, the constructs, polypeptides and/orISVDs of the invention may be formulated as a pharmaceutical preparationor composition comprising at least one construct, polypeptide and/orISVD of the invention and at least one pharmaceutically acceptablecarrier, diluent or excipient and/or adjuvant, and optionally one ormore pharmaceutically active polypeptides and/or compounds. By means ofnon-limiting examples, such a formulation may be in a form suitable fororal administration, for parenteral administration (such as byintravenous, intramuscular or subcutaneous injection or intravenousinfusion), for topical administration, for administration by inhalation,by a skin patch, by an implant, by a suppository, etc., wherein theparenteral administration is preferred. Such suitable administrationforms—which may be solid, semi-solid or liquid, depending on the mannerof administration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein. Such a pharmaceutical preparation orcomposition will generally be referred to herein as a “pharmaceuticalcomposition”.

As exemplary excipients, disintegrators, binders, fillers, andlubricants may be mentioned. Examples of disintegrators includeagar-agar, algins, calcium carbonate, cellulose, colloid silicondioxide, gums, magnesium aluminium silicate, methylcellulose, andstarch. Examples of binders include micro-crystalline cellulose,hydroxymethyl cellulose, hydroxypropylcellulose, andpolyvinylpyrrolidone. Examples of fillers include calcium carbonate,calcium phosphate, tribasic calcium sulfate, calciumcarboxymethylcellulose, cellulose, dextrin, dextrose, fructose,lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol,maltodextrins, maltose, sorbitol, starch, sucrose, sugar, and xylitol.Examples of lubricants include agar, ethyl oleate, ethyl laureate,glycerin, glyceryl palmitostearate, hydrogenated vegetable oil,magnesium oxide, stearates, mannitol, poloxamer, glycols, sodiumbenzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc.Usual stabilizers, preservatives, wetting and emulsifying agents,consistency-improving agents, flavour-improving agents, salts forvarying the osmotic pressure, buffer substances, solubilizers, diluents,emollients, colorants and masking agents and antioxidants come intoconsideration as pharmaceutical adjuvants.

Suitable carriers include but are not limited to magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatine, tragacanth, methylcellulose, sodium carboxymethyl-cellulose, alow melting-point wax, cocoa butter, water, alcohols, polyols, glycerol,vegetable oils and the like.

Generally, the constructs, polypeptides, and/or ISVDs of the inventioncan be formulated and administered in any suitable manner known per se.Reference is for example made to the general background art cited above(and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO04/041867 and WO 08/020079) as well as to the standard handbooks, suchas Remington's Pharmaceutical Sciences, 18^(th) Ed., Mack PublishingCompany, USA (1990), Remington, the Science and Practice of Pharmacy,21st Edition, Lippincott Williams and Wilkins (2005); or the Handbook ofTherapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see forexample pages 252-255).

In a particular aspect, the invention relates to a pharmaceuticalcomposition that comprises a construct, polypeptide, ISVD or nucleicacid according to the invention, and which further comprises at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally comprises one or more further pharmaceuticallyactive polypeptides and/or compounds.

The constructs, polypeptides, and/or ISVDs of the invention may beformulated and administered in any manner known per se for conventionalantibodies and antibody fragments (including ScFv's and diabodies) andother pharmaceutically active proteins. Such formulations and methodsfor preparing the same will be clear to the skilled person, and forexample include preparations preferable for suitable for parenteraladministration (e.g. intravenous, intraperitoneal, subcutaneous,intramuscular, intraluminal, intra-arterial, intrathecal intranasal orintrabronchial administration) but also for topical (i.e., transdermalor intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020079. Usually, aqueous solutions or suspensions willbe preferred.

The constructs, polypeptides, and/or ISVDs of the invention can also beadministered using methods of delivery known from gene therapy, see,e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference forits gene therapy delivery methods. Using a gene therapy method ofdelivery, primary cells transfected with the gene encoding a construct,polypeptide, and/or ISVD of the invention can additionally betransfected with tissue specific promoters to target specific organs,tissue, grafts, tumors, or cells and can additionally be transfectedwith signal and stabilization sequences for subcellularly localizedexpression.

According to further aspects of the invention, the polypeptide of theinvention may be used in additional applications in vivo and in vitro.For example, polypeptides of the invention may be employed fordiagnostic purposes, e.g. in assays designed to detect and/or quantifythe presence of ADAMTS5 and/or to purify ADAMTS5. Polypeptides may alsobe tested in animal models of particular diseases and for conductingtoxicology, safety and dosage studies.

Finally, the invention relates to a kit comprising at least onepolypeptide according to the invention, at least one nucleic acidsequence encoding said components, the vector or vector system of theinvention, and/or a host cell according to the invention. It iscontemplated that the kit may be offered in different forms, e.g. as adiagnostic kit.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

Sequences are disclosed in the main body of the description and in aseparate sequence listing according to WIPO standard ST.25. A SEQ IDspecified with a specific number should be the same in the main body ofthe description and in the separate sequence listing. By way of exampleSEQ ID no.: 1 should define the same sequence in both, the main body ofthe description and in the separate sequence listing. Should there be adiscrepancy between a sequence definition in the main body of thedescription and the separate sequence listing (if e.g. SEQ ID no.: 1 inthe main body of the description erroneously corresponds to SEQ ID no.:2 in the separate sequence listing) then a reference to a specificsequence in the application, in particular of specific embodiments, isto be understood as a reference to the sequence in the main body of theapplication and not to the separate sequence listing. In other words adiscrepancy between a sequence definition/designation in the main bodyof the description and the separate sequence listing is to be resolvedby correcting the separate sequence listing to the sequences and theirdesignation disclosed in the main body of the application which includesthe description, examples, figures and claims.

EXAMPLES Example 1 Generation of Recombinant ADAMTS5 Protein fromDifferent Species

Various formats of bovine, rat, guinea pig, mouse and cynomolgus monkeyrecombinant ADAMTS5 protein were generated in house via the HEK293Flp-In™ expression system using FuGENE® HD transfection reagent(Promega). Two days post-transfection, selection medium containing 100μg/ml HygromycinB was added to the cells to select for stably expressingcells.

Stably expressing cells were expanded. Conditioned medium containingsecreted ADAMTS5 were harvested every day from the cells, purified byHisTrap chromatography and followed by a buffer exchange in 50 mM Hepesbuffer pH 7.5. The purity of the protein was confirmed by SDS-PAGE.

Example 2 Immunization of Llamas with ADAMTS5 Protein, Cloning of theHeavy Chain-Only Antibody Fragment Repertoires and Preparation of Phage2.1 Immunizations

After approval of the Ethical Committee of the faculty of VeterinaryMedicine (University Ghent, Belgium), 3 llamas were immunized withrecombinant human ADAMTS5 Protein (R&D Systems, Minneapolis, US; cat#2198-AD).

2.2 Cloning of Heavy Chain-Only Antibody Fragment Repertoires andPreparation of Phage

Following the final immunogen injection, blood samples were collected.From these blood samples, peripheral blood mononuclear cells (PBMCs)were prepared using Ficoll-Hypaque according to the manufacturer'sinstructions (Amersham Biosciences, Piscataway, N.J., US). From thePBMCs, total RNA was extracted and used as starting material for RT-PCRto amplify the VHH/Nanobody-encoding DNA segments, essentially asdescribed in WO 05/044858. Subsequently, phages were prepared accordingto standard protocols (see for example the prior art and applicationsfiled by Ablynx N.V. cited herein.) and stored after filtersterilization at 4° C. for further use.

Example 3 Selection of Human ADAMTS5 Specific VHHs Via Phage Display

VHH repertoires obtained from all llamas and cloned in phage librarieswere used to select for human ADAMTS5 binding Nanobodies. Recombinanthuman ADAMTS5 protein (R&D Systems, Minneapolis, US; cat #2198-AD) wasimmobilized at 5 μg/ml on a Maxisorp plate (Nunc, Wiesbaden, Germany),next to a negative control of 0 μg/ml antigen. Following incubation withthe phage libraries and extensive washing, bound phages are eluted withtrypsin (1 mg/mL), and used to infect E. coli cells. Infected E. colicells were either used to prepare phage for the next selection round orplated on agar plates for analysis. In addition, synthetic librarieswere used in three consecutive selection rounds.

Outputs of all selection rounds were analyzed for enrichment factor,which is the number of phages present in eluate relative to controls.The best selection conditions were chosen for further analysis.

In order to screen a selection output for specific binders, singlecolonies were picked from the agar plates and grown in 1 mL 96-deep-wellplates. LacZ-controlled VHH expression was induced by addition of IPTG.Periplasmic extracts were prepared according to standard protocols (seefor example the prior art and applications filed by Ablynx N.V. citedherein.)

Example 4 Screening of Periplasmic Extracts for Functional BlockingNanobodies

In a first step, periplasmic extracts were tested for binding torecombinant human ADAMTS5 by binding ELISA. In brief, recombinant humanADAMTS5 (R&D Systems, Minneapolis, US; cat #2198-AD) at a concentrationof 1 μg/ml was coated on 384-well MaxiSorp plates (Nunc, Wiesbaden,Germany). Wells were blocked with a casein solution (1%). After additionof a 10-fold dilution of the periplasmic extracts, Nanobody binding wasdetected using a mouse anti-Flag-HRP conjugate (Sigma, St. Louis, US)and a subsequent enzymatic reaction in the presence of the substrateesTMB (3,3′,5,5′-tetramentylbenzidine) (SDT, Brussels, Belgium). Clonesshowing ELISA signals higher than the sum of the average signal of theirrelevant Nanobody controls and 3 times the standard deviation of theirrelevant Nanobody controls were considered to encode positive humanADAMTS5 binding Nanobodies.

To identify Nanobodies which are able to prevent ADAMTS5 mediatedcleavage of Aggrecan, clones were tested in a FRET-based human ADAMTS5enzymatic assay, using 50 mM HEPES (pH 7.5), 100 mM NaCl, 5M CaCl₂*2H₂O,0.1% CHAPS, 5% glycerol as assay buffer. In brief, periplasmic extractscontaining ADAMTS5 binding Nanobodies were incubated in a 384-wellOptiPlate (PerkinElmer, Waltham, Mass., US) with 10 μl of 25 μM quenchedfluorogenic human peptide substrate (Abz-TEGEARGSVI-Dap(Dnp)-KK-NH2,Anaspec, Serain Belgium, cat #60431-1) and 10 μl of 25 nM of humanADAMTS5 (R&D Systems, Minneapolis, US; cat #2198-AD). The ability of theNanobodies to prevent ADAMTS5 mediated cleavage was monitored everyminute for 2 hours on a Tecan Infinite M1000 plate reader. Data wereanalysed with Graphpad Prism software. From the enzyme progressioncurve, the initial velocity for the negative control (v₀) wasdetermined, as well as the velocity for every Nanobody clone in theplate (v_(i)) and the background signal (S_(b)), Using the formula100*(1−(v₁−S_(b))/(v₀−S_(b))), the percentage inhibition was calculated.

Periplasmic extract containing irrelevant Nanobody was used as negativecontrol. Periplasmic extracts containing ADAMTS5 Nanobodies which wereable to decrease the fluorescence signal with more than 50% relative tothe signal of the negative control were considered as inhibitoryNanobodies. The DNA sequence of the positive clones was subsequentlydetermined.

A summary of the periplasmic extract screening data is given in Table4.1. The amino acid sequences of the anti-ADAMTS5 Nanobodies are shownin Table A-1.

TABLE 4.1 Screening results of periplasmic extracts containinganti-ADAMTS5 Nanobodies binding ELISA FRET assay sequence ID (OD 450 nm)vi (U/min) % inh 2A02 1.646 2 91 2A12 1.745 −1 104 2C10 1.627 2 90 2D071.551 −1 104 2D12 1.354 2 90 2F03 1.842 −1 103 2G01 1.710 10 55 3B022.570 −1 105 3B03 2.295 2 93 3D01 2.573 −2 111 3D02 2.581 −2 111 7B110.368 2 90 9A05 ND −3 113 9D03 ND −2 110 9D09 ND 0 102 9D10 ND −2 11111B06 ND −1 106 13E02 ND 0 99 3F04 2.555 19 8

After cloning and sequencing, various families were identified, whichconsisted of clones with differences in CDRs, but which displayedsimilar binding and inhibition characteristics.

After cloning and sequencing, Nanobody 02A12 was identified as a familymember of clone 09D03 (cf. Tables A-1 and A-2). The sequence variabilityof CDR regions is depicted in the Tables 4.1A, 4.1B and 4.1C below. Theamino acid sequences of the CDRs of clone 09D03 were used as referenceagainst which the CDRs of the family members were compared (CDR1 startsat Kabat position 26, CDR1 starts at Kabat position 50, and CDR3 startsat Kabat position 95).

TABLE 4.1A (09D03 CDR1) 09D03 CDR1* Kabat 26 27 28 29 30 31 32 33 34 35numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G S A V S V NA M A sequence mutations R T F S Y G *Up to 6 CDR1 mutations in oneclone (SEQ ID NO: 22)

TABLE 4.1B (09D03 CDR21) 09D03 CDR2* Kabat 50 51 52 53 54 55 56 57 58 59numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G I S R S A GR T Y sequence mutations *Up to 0 CDR2 mutations in one? clone (SEQ IDNO: 36)

TABLE 4.1C (09D03 CDR3) 09D03 CDR3* Kabat 95 96 97 98 99 100 100anumbering absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 numberingwildtype D L D P N R I F S R D E A A Y sequence mutations . . . . *Up to0 CDR mutations in one clone (SEQ ID NO: 54)

After cloning and sequencing Clone 09A05 was identified as a familymember of clone 03B02 (cf. Tables A-1 and A-2). The sequence variabilityof CDR regions is depicted in the Tables 4.1D, 4.1E and 4.1F below. Theamino acid sequences of the CDRs of clone 03B02 were used as referenceagainst which the CDRs of the family members were compared (CDR1 startsat Kabat position 26, CDR1 starts at Kabat position 50, and CDR3 startsat Kabat position 95).

TABLE 4.1D (03B02 CDR1) 03602 CDR1* Kabat 26 27 28 29 30 31 32 33 34 35numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype R R T I S S GT M G sequence mutations *Up to 0 CDR1 mutations in one clone (SEQ IDNO: 33)

TABLE 4.1E (03B02 CDR2) 03B02 CDR2* Kabat 50 51 52 53 54 55 56 57 58 59numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype A I R W S S GM P Y sequence mutations I T F *Up to 3 CDR2 mutations in one clone (SEQID NO: 50)

TABLE 4.1F (03B02 CDR3) 03B02 CDR3* Kabat 95 96 97 98 99 100 100anumbering absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 numberingwildtype D R S A F R D P S F D V N Y E Y sequence mutations . . . L E *Up to 2 CDR mutations in one clone (SEQ ID NO: 68)

After cloning and sequencing clones 03D02 and 02C10 were identified asfamily members of clone 03D01 (cf. Tables A-1 and A-2). The sequencevariability of CDR regions is depicted in the Tables 4.1G, 4.1H and 4.11below. The amino acid sequences of the CDRs of clone 03D01 were used asreference against which the CDRs of the family members were compared(CDR1 starts at Kabat position 26, CDR1 starts at Kabat position 50, andCDR3 starts at Kabat position 95).

TABLE 4.1G (03D01 CDR1) 03D01 CDR1* Kabat 26 27 28 29 30 31 32 33 34 35numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype G F T F S P YY M G sequence mutations *Up to 0 CDR1 mutations in one clone (SEQ IDNO: 28)

TABLE 4.1H (03D01 CDR2) 03D01 CDR2* Kabat 50 51 52 53 54 55 56 57 58 59numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering wildtype A I S R S R GT T Y sequence mutations T W I L *Up to 3 CDR2 mutations in one clone(SEQ ID NO: 44)

TABLE 4.1I (03D01 CDR3) 03D01 CDR3* Kabat 95 96 97 98 99 100 100anumbering absolute 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 numberingwildtype G R S P G D P S R T Y L Y D Y sequence mutations S . . . E *Upto 1 CDR mutation in one clone (SEQ ID NO: 62)

The above results demonstrate that some amino acid variation ispermissible, while retaining binding and inhibition characteristics.Notably, binding to ADAMTS5 is not a predictor for inhibition of ADAMTS5activity. For instance, clone 3F04 is a potent binder but a weakinhibitor.

Example 5 Characterization of Purified Monovalent ADAMTS5 Nanobodies

Clones selected from the screening described in Example 4 were furthercharacterized. Upon transformation in E. coli (TG-1) of selectedNanobodies, expression was induced by addition of 1 M IPTG and allowedto continue for 4 hours at 37° C. After spinning the cell cultures,periplasmic extracts were prepared by freeze-thawing the pelletsfollowed by centrifugation. These extracts were then used as startingmaterial for purification via IMAC on HisTrap FF crude 1 ml columns (GEhealthcare, Buckinghamshire, United Kingdom) followed by desalting viaZeba spin columns (Pierce, Rockford, Ill., USA) resulting in at least95% purity as assessed via SDS-PAGE.

5.1 Evaluation of ADAMTS5 Blocking Nanobodies in Enzymatic ADAMTS5Assays

The ability of Nanobodies to prevent ADAMTS5 mediated cleavage ofAggrecan was confirmed in a FRET-based enzymatic assay, essentially asdescribed in Example 4. Data were analysed with Graphpad Prism software.From the enzyme progression curve, the initial velocity was determined(v_(i)) and these values were plotted as a function of inhibitorconcentrations and IC₅₀ values were calculated. In essence, the humanFRET assay with purified Nanobodies confirmed the results described inExample 4 with the periplasmic extracts. The IC₅₀s ranged between 1.8E⁻⁰⁹ M to 3.7 E⁻⁰⁸ M. Notably, although the conventional mAb 12F4 had asomewhat better IC₅₀ of 1.0 E⁻⁰⁹ M than the monovalent Nanobodies, allNanobodies showed better efficacy (data not shown).

Next to the FRET-based assay, an AlphaLSA (Perkin Elmer, Waltham, Mass.,US) based human ADAMTS5 assay with a biotinylated 43-mer Aggrecanoligopeptide as substrate was performed. Upon ADAMTS5 cleavage of thissubstrate, a biotinylated ARGSV neo epitope product is released, whichcan be detected by streptavidin-AlphaScreen donor beads and an anti-neoepitope (“ARGSV”) antibody captured on anti-mouse IgG-coated AlphaLSAacceptor beads, resulting in the generation of a luminescenceAlphaScreen signal upon laser excitation.

To determine the inhibitory potential of the Nanobodies, serialdilutions of purified Nanobodies (together with a positive control and anegative irrelevant Nanobody control) were incubated with human ADAMTS5(R&D Systems, Minneapolis, US; cat #2198-AD) for 15 minutes at roomtemperature in a 384-well Optiplate (Perkin Elmer, Waltham, Mass., US).Following addition of a biotinylated 43-mer Aggrecan oligopeptidesubstrate (Biosyntan, Berlin, Germany) and incubation of 3 hours at 37°C. the reaction was stopped. The first detection step was performed bythe addition of 5 μl detection solution 1 comprising Aggrecan antibodyagainst the neo-epitope “ARGSV”, mouse BC3 mAb (MDBioproducts, Egg,Switzerland) and Streptavidin AlphaScreen donor beads (Perkin Elmer).After 1 h incubation at room temperature, 5 μl of a second detectionsolution was added, comprising the anti-mouse AlphaLISA acceptor beads(Perkin Elmer). The plates were incubated for 2 hours at roomtemperature in the dark. Measurement was performed by reading the plateson the Envision Multi label Plate reader (Perkin Elmer, Waltham, Mass.,US).

To determine the ability of the Nanobodies to prevent ADAMTS5 mediatedcleavage of the substrate, the decrease in signal was analysed infunction of Nanobody concentration and ICS® values were calculated.

The results are summarized in Table 5.1.

TABLE 5.1 Potency (IC₅₀) and % inhibition of ADAMTS5 for Nanobodies andreference compounds in human AlphaLISA enzymatic assay. Human AlphaLISACompound ID IC50 [M] % inhibition mAb 12F4 H4L0 6.5E−11 100 2F03 7.5E−11100 11B06 1.0E−10 100 9D03 1.1E−10 100 2A12 1.5E−10 100 2D07 1.9E−10 1003D02 2.2E−10 100 3B02 3.0E−10 100 9A05 3.2E−10 100 3D01 3.9E−10 96 2C104,5E−10 100 rhTIMP3 6.1E−10 100 2D12 6.9E−10 100 9D10 9.7E−10 100 13E022,2E−09 100 9D09 2.6E−09 100 7B11 3.6E−09 100 2A02 4.3E−09 99 3B031.2E−08 100 MSC2310852A 1.4E−07 99

In the AlphaLISA assay, at least three monovalent Nanobodies (2F03,11806 and 9D03) show similar IC₅₀ values than the conventional mAb 12F4,while the remainder has higher values. However, all Nanobodies show anear 100% inhibition. Remarkably, Nanobody 3B03, which has an IC₅₀ valueat least a factor 100 higher than mAb 12F04 still has 100% inhibition.

5.2 Evaluation of ADAMTS5 Blocking Nanobodies in Bovine Explant Assay

Evaluation of the ability of the Nanobodies to block cartilagedegradation in an ex vivo assay, in which the substrate is presented ina condition closer to the physiological condition compared tobiochemical assays, a bovine explant assay was performed. In brief,bovine cartilage explant chips (diameter 4 mm) were prepared freshlyfrom cow knee joints and incubated in 96-well plates in presence ofIL-la to induce cartilage degradation. As a measure ofcartilage/Aggrecan degradation, the release of GAG was detected in thesupernatant after 5 days of incubation (37° C., 7.5% CO₂) via themetachromatic dye 1,9 dimethylmethylene blue (emission at 633 nm).Chondroitin sulphate was included as assay standard. Efficacy wasdefined by means of the IL-1α-induced controls without compound (0%) andin presence of MSC2310852A (100% effect).

Results are summarized in Table 5.2.

TABLE 5.2 IC₅₀ values from monovalent Nanobodies in the bovine explantassay. ID IC50 [M] Experiment ID* mAb 12F4 H4L0  3.2E−06 exp A 2F03 1.4E−08 exp A 11B06 4.80E−08 exp C 9D03  5.9E−08/2.7E−07 exp G/exp F2A12 2.40E−08 exp C 02D07 3.30E−08 exp C 3D02 8.80E−07 exp F 3B021.40E−07 exp D 9A05 5.10E−07 exp F 3D01  2.2E−08/1.3E−07 exp D/exp G2D12 8.00E−07 exp D 2A02 2.40E−06 exp D *results can only be comparedwithin 1 assay using the same explant, with identical experiment ID

5.3 Epitope Binning

An SPR-based “sandwich assay” on a Biacore T100 instrument was performedto group the ADAMTS5 Nanobodies into different epitope bins. To thisend, each anti-ADAMTS5 Nanobody was immobilized on a CM5 sensor chip viaamine coupling using EDC and NHS chemistry. After a capturing step of100 nM ADAMTS5, purified Nanobodies at a concentration of 1 μM wereinjected, each in a single cycle and without regeneration, with asurface contact time of 120 seconds and a flow rate of 45 μL/minute.Curves were processed with Biacore T100 Evaluation software andevaluated for additional binding to the captured ADAMTS5 (different bin)or not (same bin).

Nanobodies could be divided in two groups: one large bin (“bin 1”),which comprises all inhibitory Nanobodies having similar or overlappingepitopes, and a second group comprising the binding, non-functionalNanobody 3F04 which recognizes a different epitope on ADAMTS5 (“bin 2”).

Table 5.3 Summarizes the Binding Epitopes of the Tested Nanobodies.

TABLE 5.3 epitope bins of the anti-ADAMTS5 Nanobodies bin 1 2A02 2A122C10 2D07 2D12 2F03 2G01 3B02 3B03 3D01 3D02 7B11 9A05 9D03 9D09 9D1011B06 13E02 bin 2 3F04

5.4 Species Cross Reactivity

Species cross-reactivity was initially evaluated via SPR-based off-rateanalysis on a Biacore T100 instrument. Nanobodies were tested forbinding to human, cynomolgus monkey (“cyno”), guinea pig, mouse, andbovine ADAMTS5. To this end, recombinant ADAMTS5 was immobilized onto aCM5 chip via amine coupling using EDC and NHS. Purified Nanobodies wereinjected for 2 minutes at a concentration of 100 nM and allowed todissociate for 15 min at a flow rate of 45 ul/min. Off-rate for eachindividual Nanobody was determined by fitting a 1:1 interaction model(Langmuir model) onto the individual dissociation curves using the BIAEvaluation software. As a reference, off-rates on human ADAMTS5 weredetermined in each experiment.

The results are summarized in Table 5.4A.

TABLE 5.4A Overview of species cross reactivity data of monovalentanti-ADAMTS5 Nanobodies SPR based off-rate, kd (Vs) on ADAMTSS NanobodyExperiment 1 Experiment 2 Experiment 3 ID Human Cyno Guinea pig HumanMouse Human Bovine 2F03 3.3E−05 2.2E−05 3.4E−05 6.5E−05 2.9E−04 9.7E−054.3E−05 11806 1.7E−04 1.4E−04 1.7E−04 2.7E−04 5.3E−04 3.8E−04 2.4E−049D03 9.8E−04 9.2E−04 1.0E−03 1.2E−03 3.7E−03 1.4E−03 9.9E−04 2A121.4E−04 1.0E−04 1.2E−04 2.3E−04 4.4E−04 3.1E−04 1.7E−04 2D07 1,1E−039.8E−04 1.1E−03 9.4E−04 1.8E−03 1.0E−03 6.2E−04 3B02 7.3E−04 7.4E−041.0E−03 8.5E−04 2.0E−03 1.2E−03 1.1E−03 3D01 2,3E−04 2.0E−04 2.7E−043.2E−04 6.4E−04 4.6E−04 3.5E−04

For each Nanobody tested, similar off-rates were observed for all testedspecies.

In addition, cross-reactivity for cynomolgus monkey and guinea pigADAMTS5 was also addressed by potency determination using AlphaLISA,essentially as described in Example 5.1, but using a biotinylatedcynomolgus 43-Aggrecan oligopeptide substrate and a biotinylated guineapig 43-Aggrecan oligopeptide substrate. To determine the ability of theNanobodies to prevent ADAMTS5 mediated cleavage of the substrate, thedecrease in signal was analysed in function of Nanobody concentrationand IC₅₀ values were calculated.

The resulting potencies are summarized in Table 5.4B.

TABLE 5.4B Overview of species cross reactivity data of monovalentanti-ADAMTS5 Nanobodies Potency (IC50, M) Nanobody AlphaLISA ID HumanCyno Guinea pig 2F03 7.5E−11 8.1E−11 3.0E−10 11B06 1.0E−10 1.2E−103.5E−10 9D03 1.1E−10 1.2E−10 1.3E−10 2A12 1.5E−10 1.9E−10 5.3E−10 2D071.9E−10 1.5E−10 3.6E−10 3B02 3.0E−10 1.9E−10 4.6E−10 3D01 3.9E−102.2E−10 3.5E−10

All Nanobodies and the control compounds blocked human and cyno ADAMTS5with similar potencies. In the guinea pig AlphaLISA, the most potentNanobodies showed slightly lower potencies than in the human AlphaLISA,but this is most likely the result of lower assay sensitivity due toincreased enzyme concentration.

Rat cross-reactivity of Nanobodies 2A12, 2F03, 093 and 049 was evaluatedby means of an SPR-based affinity determination using Biacore T100. Forreferencing, affinity for human ADAMTS5 was also determined. Thereto,rat and human ADAMTS5 were immobilized onto a CM5 chip via aminecoupling, using EDC and NHS chemistry. Purified Nanobodies were injectedfor 2 minutes at different concentrations (between 1 and 1000 nM) andallowed to dissociate for 25 min at a flow rate of 45 μl/min. Thekinetic constants were calculated from the sensorgrams using theBIAEvaluation software (1:1 interaction).

As presented in Table 5.4C, similar affinities on human ADAMTS5 and ratADAMTS5 were obtained for all tested Nanobodies. The affinity of thehalf-life extended Nanobody 069 (cf. infra) was similar to the affinityof its monovalent counterpart (which is Nanobody 049), indicating thatthe addition of ALB11 at the C-terminus has no effect on the affinity.The affinity of half-life extended biparatopic Nanobody 130 was on bothspecies higher (15-fold on human ADAMTS5 and 10-fold on rat ADAMTS5)compared to the affinity of half-life extended Nanobody 069 (Table5.4C).

TABLE 5.4C overview of affinities on human ADAMTS5 and rat ADAMTS5 KD(nM) KD (nM) human rat ID Description ADAMTS5 ADAMTS5 2A12 1.9 1 2F031.6 0.7 093 3F04 (N100_(f)Q) 4.9 5.3 049 11B06 (N52S) 3.5 1.7 069049-35GS-ALB11 2.7 1.3 130 049-35GS-093-35GS-ALB11 0.18 0.13

5.5 Inhibition of MMP-1 and MMP-14 Activity

To confirm the selectivity of the Nanobodies for ADAMTS5, inhibition ofthe enzyme activity of MMP-1 and MMP-14 was evaluated with FRET-basedassays with the respective enzymes. In brief, activated human MMP-1 orMMP-14 was incubated for 30 minutes at room temperature with 10 μl ofdilution series of Nanobody. After incubation, 20 μl of respectively 5μM or 2.5 μM fluorogenic peptide substrate (Mca-PLGL-Dpa-AR-NH2Fluorogenic MMP Substrate (R&D Systems cat # ES001)) was added. Theability of the Nanobodies to prevent MMP-1 and MMP-14 mediated cleavagewas monitored every minute for 2 hours at 37° C. on a Tecan InfiniteM1000 plate reader.

The resulting titration curves are shown in FIG. 1.

Whereas the natural inhibitors TIMP2 and TIMP3 inhibited MMP-1 andMMP-14 activity, none of the tested Nanobodies showed inhibition.

5.6 Inhibition of Human ADAMTS4 Activity

To evaluate inhibition of human ADAMTS4, an assay similar to the humanADAMTS5 AlphaLISA was carried out, essentially as described in Example5.1, but using human ADAMTS4 (R&D Systems, Minneapolis, US; cat#4307-AD). To determine the ability of the Nanobodies to prevent humanADAMTS4 mediated cleavage of the substrate, the decrease in signal wasanalysed in function of Nanobody concentration and IC₅₀ values werecalculated.

The results are shown in FIG. 2.

Whereas the small molecule MSC2310852A inhibited human ADAMTS4 activity,none of the tested Nanobodies or mAb 12F4 showed inhibitory activity(see Error! Reference source not found.). The monoclonal antibody 12F4(H4L0) was described to be selective over ADAMTS4 in WO 2011/002968.

Example 6 Formatting of Nanobodies 6.1 Knock Out of N-GlycosylationMotifs

N-glycosylation motifs present in Nanobody 11B06 (position N52) andNanobody 3F04 (position N110f) were knocked out prior to formatting. Thesites were randomized by means of an NNK codon library. Since thesepositions are located in the CDRs, mutation of the motif may potentiallyinfluence binding characteristics. Accordingly, the libraries werescreened for possible substitutions which did not affect the bindingproperty by SPR based off-rate analysis on human ADAMTS5 using a BiacoreT100 instrument, essentially as described in Example 5.4.

Surprisingly, substituting amino acid N52 by Serine in Nanobody 11B06and amino acid N100_(F) by Glutamine in Nanobody 3F04 did not affectbinding (data not shown).

Hence, as building blocks for further formatting, Nanobodies 049 (=11B06(N52S)) and 093 (3F04 (N100_(F)Q)) were used to represent Nanobodies11B06 and 3F04, respectively (see Table A-1).

6.2 Generation of Formatted Nanobodies

For the generation of half-life extended Nanobody constructs which blockhuman ADAMTS5 aggrecanase activity, Nanobodies 2A12, 2D07, 2F03, 049,9D03 and 3B02 were fused to an anti-Human serum albumin (HSA)-NanobodyALB11 (see Table 6.3). In addition, ALB11 half-life extended constructswere also generated from a combination of two Nanobodies from differentbinding epitopes (a combination of a bin 1 member and a bin 2 member).The formatted Nanobody constructs were expressed in Pichia pastoris,secreted into the cultivation medium and affinity purified on PorosMabCaptureA Protein A beads (Applied Biosystems, Bleiswijk, Netherlandscat #4374729). The integrity of the Nanobody constructs was confirmed.

6.3 Potency in AlphaLISA in Absence and Presence of HSA

The formatted Nanobody constructs were tested in the AlphaLSA, asdescribed in Example 5.1. Additionally, the influence of HSA binding onNanobody potency was addressed by performing the experiment in presenceof an excess of HSA (4.2 μM final concentration).

The IC₅₀ values of all half-life extended Nanobody constructs are listedin Table 6.3.

TABLE 6.3 Potencies of formatted Nanobody constructs (+/− HSA) asdetermined in AlphaLISA Nanobody IC50 (M) ID Description − HSA + HSA 0042A12-35GS-ALB11 1.3E−10 1.1E−10 005 2D07-35GS-ALB11 2.9E−10 3.4E−10 0062F03-35GS-ALB11 7.0E−11 9.2E−11 069 049-356S-ALB11 6.9E−11 9.8E−11 0709D03-35GS-ALB11 4.1E−10 6.0E−10 071 3B02-35GS-ALB11 2.8E−10 4.1E−10 1292F03-35GS-093*-35GS-ALB11 1.1E−10 ND 130 049²*-35GS-093-35GS-ALB119.7E−11 ND 131 9D03-35GS-093-35GS-ALB11 8.1E−31 ND Nanobody IC50 (M) ID− HSA 2A12 1.5E−10 2D07 1.9E−10 2F03 7.5E−11 049 1.0E−10 9D03 1.1E−103B02 3.0E−10 ND = not determined; 093* = 3F04 (N100_(f)Q); 049²* = 11B06(N52S)

The results indicate that neither ALB11 formatting nor HSA bindingaffects the potency of the Nanobody constructs.

6.4 Binding to HSA

The affinity of the ALB11 Nanobody to HSA was determined via SPR on aBiacore T100. To this end, HSA was immobilized onto a CM5 chip via aminecoupling and processed essentially as described in Example 5.3 As areference, the affinity of monovalent ALB11 was also determined, whichwas 3.2 nM.

The results are summarized in Table 6.4.

TABLE 6.4 Affinity of HLE Nanobody constructs for RSA ID KD (nM) fromFNb* FNb-ALB11 FNb-093³*-ALB11 FNb-ALB11-093 2F03 46 66 52 049²* 40 5540 2A12 27 ND ND ND = not determined.; *FNb = functional Nanobody; 049²*= 11B06 (N52S); 093³* = 3F04 (N100_(f)Q)

All half-life extended ADAMTS5 Nanobody constructs had similaraffinities (see Table 6.4), which was lower than the affinity ofmonovalent ALB11 for HSA.

6.5 Affinity Determination for Human ADAMTS5 with KinExA

The affinity of the formatted Nanobody construct 069 (049-35GS-ALB11)and Nanobody construct 130 (049-35GS-093-ALB11)(see also Table 6.5) weredetermined in solution with a kinetic exclusion assay on a KinExA3000instrument (Sapidyne, Boise, USA). To this end, human ADAMTS5 wascoupled to PMMA beads according to the manufacturer's instructions,described in detail by Darling and Brault (2004 Assay Drug Dev Technol.2:647-57). A fixed concentration of Nanobody construct (50 pM Nanobodyconstruct 069 or 5 pM Nanobody construct 130) was added to a dilutionseries of human ADAMTS5 ranging from 2 nM-0.2 pM and incubated at roomtemperature for 16 hours till equilibrium was reached. Subsequently,mixtures were injected via KinExA's autosampler over a column packedwith human ADAMTS5-conjugated PMMA beads to capture free Nanobodyconstruct on the beads and detect with Alexa647-labeled HSA. Percentfree Nanobody construct was plotted as a function of titrated humanADAMTS5 and fitted using the KinExA Pro Software v3.2.6.

The results are shown in Table 6.5.

TABLE 6.5 Affinities of 2 Nanobody constructs for binding to humanADAMTS5 as determined via KinExA Nanobody Description KD (pM) 069049*-35GS-ALB11 20.4 130 049-35GS-093*-ALB11 1.2 049* = 11B06 (N52S);093²* = 3F04 (N100_(f)Q)

A circa 20 times higher affinity is observed for Nanobody construct 130compared to Nanobody construct 069. These data confirm the avid bindingon human ADAMTS5 of Nanobody construct 130.

Example 7 Sequence Optimization (SO) of Nanobodies

Exemplary Nanobody 2F03, Nanobody 049 (11B06 (N52S)) and Nanobody 093(3F04 (N100_(F)Q)) were subjected to a sequence optimisation process.Sequence optimisation is a process in which a parental Nanobody sequenceis mutated and this process covers the humanization (i) of the Nanobodyand knocks-out post-translational modifications (ii) as well as epitopesfor potential pre-existing antibodies (iii). Nanobody ALB11 was alsomutated to knock out epitopes for potential pre-existing antibodies.

-   (i) for humanization purposes the parental Nanobody sequence is    mutated to yield a Nanobody sequence which is more identical to the    human IGHV3-IGHJ germline consensus sequence. Specific amino acids    in the framework regions (with the exception of the so-called    hallmark residues) that differ between the Nanobody and the human    IGHV3-IGHJ germline consensus are altered to the human counterpart    in such a way that the protein structure, activity and stability are    kept intact. A handful of hallmark residues are known to be critical    for the stability, activity and affinity of the Nanobody and are    therefore not mutated.-   (ii) the amino acids present in the CDRs and for which there is    experimental evidence that they are sensitive to post-translational    modifications (PTM) are altered in such a way that the PTM site is    inactivated while the protein structure, activity and stability are    kept intact.-   (iii) the sequence of the Nanobody is optimised, without affecting    protein structure, activity and stability, to minimise binding of    any naturally occurring pre-existing antibodies and reduce the    potential to evoke a treatment-emergent immunogenicity response.

For the generation of sequence optimised formatted Nanobody construct581 and Nanobody construct 579, respective building blocks wereconnected via 35GS linkers. The resulted in Nanobody construct 581(2F3^(SO)-35GS linker-Alb11) and Nanobody construct 579 (2F3^(SO)-35GSlinker-093^(SO)-35GS linker-Alb11), respectively (cf. Table A-1). Theconstructs were produced in Pichia pastoris as tagless proteins andpurified via Protein A affinity chromatography, followed by desalting.The integrity of the Nanobody constructs was confirmed.

7.1 Affinity for human ADAMTS5

Affinity of Nanobody construct 581 (2F3^(SO)-35GS linker-Alb11) wasdetermined using KinExA, as described in Example 6.5 with a few minoradaptations to the described protocol. A fixed concentration of 20 pM ofNanobody construct 581 was incubated together with a dilution series ofhuman ADAMTS5 (2.2-fold serial dilutions ranging between 20 nM and 0.32pM and a blank without ADAMTS5). To test for interference of albumin,HSA was added to this pre-incubation in selected experiments with humanADAMTS5, at a concentration of 100-fold its KD for Nanobody construct581. Mixtures were incubated and allowed to reach equilibrium for 24hours prior to injection via KinExA's autosampler over a column packedwith human ADAMTS5-conjugated PMMA beads. Captured Nanobody construct581 was detected using an AF647-labelled anti-Nanobody tool recognizingNanobody construct 581 (generated by Ablynx).

Results are presented in Table 7.1.

TABLE 7.1 Affinity of Nanobody construct 581 for human ADAMTS5 with andwithout HSA + or − HSA K_(D) (pM) 95% Cl on K_(D) (pM) − HSA (n = 3)3.65 (CV 20%) 2.27-5.86 + HSA (n = 3) 4.84 (CV 22%) 3.45-6.80

The results show that the affinity of Nanobody construct 581 for humanADAMTS5 is 3.65 pM without HSA and 4.84 pM with HSA.

7.2 Affinity for Serum Albumin

Affinity of the ALB11 building block in Nanobody construct 581(2F35^(SO)-35GS linker-Alb11) for binding to human, cynomolgus monkey,guinea pig, mouse and rat serum albumin (SA) was determined using SPR asdescribed in Example 5.3.

Results are shown in Table 7.2.

TABLE 7.2 Affinity (K_(D), nM) of Nanobody construct 581 for binding tohuman, cynomolgus monkey, guinea pig, mouse and rat serum albuminNanobody ID human SA cyno SA guinea Pig SA mouse SA rat SA 581 48 51 420790 >7500* *off-rate is outside detection limit: >5.0E−01 (1/s)

7.3 Pre-Ab Binding

Nanobody construct 581 (2F3^(SO)-35GS linker-Alb11) and Nanobodyconstruct 579 (2F3^(SO)-35GS linker-093^(SO)-35GS linker-Alb11) werescreened for pre-existing antibody (pre-Ab) binding from 3 relevantdonor serum sample sets (samples from healthy subjects, OA samples and abiased set of samples with known residual pre-Ab binding to otherNanobodies) via SPR technology on a ProteOn XPR36 instrument. Bloodsamples were analysed for binding to anti-ADAMTS5 Nanobody constructs,which were captured on HSA. Pre-existing antibody binding levels weredetermined after double referencing of the sensorgrams by setting reportpoints at 125 seconds (5 seconds after end of association). Analysis wasperformed with ProteOn manager 3.1 (Bio-Rad Laboratories, Inc.).

The results are shown in FIG. 3.

Both Nanobody constructs had low residual pre-Ab binding levels, withNanobody construct 581 clearly showing the least residual pre-Ab binding(cf. Error! Reference source not found.).

7.4 Functionality in Human AlphaLISA

An AlphaLISA was performed with Nanobody construct 581 (2F3^(SO)-35GSlinker-Alb11) using human ADAMTS5 essentially as described in Example5.1.

The results are shown in Table 7.4.

TABLE 7.4 Potency (pM) of Nanobody construct 581 as determined byAlphaLISA. Sample IC50 (pM) 95% Cl (pM) mAb 12F4H4L0 120 102-140 581 188157-226

The results show that the potency of Nanobody construct 581 is 188 pM.

7.5 Immunogenicity Profiling in Dendritic Cell-T Cell Assay

The relative immunogenicity was determined in a Dendritic Cell-T cellproliferation assay. In essence, Nanobodies were tested against a set of50 healthy donor cell samples containing the most abundant HLA class IIalleles, as such representing the majority of the global population. Theimmune response was assessed using T cell proliferation as a surrogatemarker for anti-drug antibody formation. Keyhole Limpet Hemocyanin (KLH)was used as a positive control. The positive control KLH led to apositive response in all of the 50 donors. None of the donors testedpositive for Nanobody construct 581 (2F3⁰-35GS linker-Alb11). Nanobodyconstruct 579 (2F3^(SO)-35GS linker-093^(SO)-35GS linker-Alb11) led to apositive response in three donors, or 6%. In the blank condition (mediumonly), 2 out of 50 donors, or 4%, were found to respond positive. Theseresults were used to set an overall response threshold of more than 3out of 50 donors corresponding to an overall response threshold of >6%.The overall immunogenic potential for the tested Nanobodies wasconsidered low as the percentage of significant, positive responses was6% (see Table 7.5).

TABLE 7.5 Result immunogenicity profiling as determined in a DC-T CellAssay # Positive Immunogenic Test product responses potential Blank 2(4%) / KLH 50 (100%) / 581 0 (0%) Low 579 3 (6%) Low Control Nb 1 (2%)Low

7.6 Binding to Human ADAMTS1

Binding to human ADAMTS1 was tested in a binding ELISA (see Error!Reference source not found. A). In brief, 18.5 nM of human ADAMTS1 (R&DSystems, Minneapolis, US; cat #2197-AD) and human ADAMTS5 (R&D Systems,Minneapolis, US; cat #2198-AD) were coated on 384-well MaxiSorp plates(Nunc, Wiesbaden, Germany). Wells were blocked with a casein solution(1%) and dilution series of Nanobody construct 581 (2F3^(SO)-35GSlinker-Alb11) and anti-human ADAMTS1 mAb (R&D Systems, Minneapolis, US;cat # MAB2197) were tested for binding on both coated proteins. Afterdetection with an HRP conjugated anti-Nanobody tool recognizing Nanobodyconstruct 581 (generated by Ablynx) and an HRP conjugated anti-mouseantibody (Abcam, Cambridge, UK) respectively, and a subsequent enzymaticreaction in the presence of the substrate esTMB(3,3′,5,5′-tetramentylbenzidine) (SDT, Brussels, Belgium), OD450 nm wasmeasured.

A dose response curve was observed for Nanobody construct 581 on humanADAMTS5 and for anti-human ADAMTS1 mAb on human ADAMTS1, showing thatNanobody construct 581 does not bind to human ADAMTS1.

7.7 Binding to Human ADAMTS4 and ADAMTS15

Binding to human ADAMTS4 and ADAMTS15 was tested in a binding ELISA (seeFIG. 4B). In brief, 1 μg/mL of human ADAMTS4 (R&D Systems, Minneapolis,US; cat #4307-AD) or human ADAMTS15 (R&D Systems, Minneapolis, US; cat#5149-AD) was coated overnight at 1 μg/mL onto 96-well Maxisorp ELISAplates (Nunc, Wiesbaden, Germany). After blocking of the plates withSuperBlock T20 (PBS) (Thermo Scientific Pierce; cat #37516) a doseresponse curve of Nanobody construct 581 (“C011400581”) or the positivecontrol anti-ADAMTS4 mAb (R&D Systems, Minneapolis, US; cat # MAB4307)or the positive control anti-ADAMTS15 mAb (R&D Systems, Minneapolis, US;cat # MAB5149) was applied on the coated plate starting at 1 μM andincubated for 1 hour at room temperature. Bound C011400581 Nanobody wasdetected with a biotinylated anti-Nanobody tool recognizing Nanobodyconstruct 581 (generated by Ablynx), while the positive control mAb'swere detected with a biotinylated goat anti-mouse pAb (Jackson ImmunoResearch; cat #115-065-062). For both streptavidin-HRP was used as asecondary detection tool (Thermo Scientific Pierce; cat #21126). Plateswere then visualized by adding sTMB) (SDT, Brussels, Belgium). Alldilutions and detection tools were prepared in assay diluent (=PBS+10%Superblock+0.05% Tween20). The colouring reaction was stopped after 2.5minutes by adding 1M HCl. Optical density was measured at 450 nm with620 nm as reference wavelength.

7.8 Species Cross-Reactivity Via KinExA Affinity Determination

Affinity of Nanobody construct 581 (2F3^(SO)-35GS linker-Alb11) towardsseveral species' ADAMTS5 was determined using KinExA, as described inExample 7.1 For all species, human ADAMTS5 conjugated PMMA beads wereprepared and use for capture of free Nanobody construct 581 as describedabove. Captured Nanobody construct 581 was detected using anAF647-labelled anti-Nanobody tool recognizing Nanobody construct 581(generated by Ablynx). The results are shown in Table 7.7

TABLE 7.7 Affinity of Nanobody construct 581 for ADAMTS5 of cynomolgusmonkey, rat, mouse and bovine Species ADAMTS5 K_(D) (pM) 95% Cl on K_(D)(pM) Cynomolgus 2.22 0.88-4.76 Monkey (n = 1) Rat (n = 1) 2.77 1.48-5.07Mouse (n = 1) 2.81  0.30-11.42 Bovine (n = 1) 7.66  3.33-19.88

Example 8 Potency in Human Explant Assay

Nanobody construct 581 (2F3^(SO)-35G5 linker-Alb11) was evaluated on theability to block cartilage degradation in an ex vivo assay, essentiallyas described in Example 5.2, but now in a human explant assay. In brief,human cartilage explant chips (diameter 4 mm) were prepared freshly fromhuman knee joints and incubated in 96-well plates in presence of 10ng/ml IL-1c to induce cartilage degradation). As a measure ofcartilage/Aggrecan degradation, the release of GAG was detected in thesupernatant after 7 days of incubation (37° C., 7.5% CO₂) via themetachromatic dye 1,9 dimethylmethylene blue (emission at 633 nm).Chondroitin sulphate was included as assay standard. Efficacy wasdefined by means of the IL-1β and OSM induced controls without compoundand in presence of MSC2310852A. CRB0017 is an anti-ADAMTS5 mAb that isto be used in a Phase I clinical trial (Rottapharm).

Results are summarized in FIG. 5 and Table 8.

TABLE 8 IC50 GAG Compound Assay [μM] 581 Human 0.0025-0.0079 581 Bovine0.035 CRB0017 Bovine 0.33

Nanobodies demonstrate high potency and 100% inhibition of cartilagedegradation (GAG loss) in human explant assay. As shown in Table 8, theNanobodies are at least 10-fold better than CRB0017 in the bovineexplant assay.

Example 9 Modulation C2M, C3M and exAGNX1 in a Bovine Co-Culture Model

The effect of Nanobody construct 581 (“581” in the figures) on GAGrelease and content, C2M (marker of collagen II degradation), C3M(marker of collagen Ill degradation) and exAGNx1 (marker for aggrecandegradation characterized the neo-epitope TEGE) in a bovine co-culturesystem was investigated. In this co-culture systems, cartilage explantsfrom a bovine knee were incubated together with the synovial membranefrom the same animal for 28 days. Highest GAG release was detected after7 days in culture (FIG. 6A). The Nanobody construct 581 was tested inthree concentrations: 1 nM, 10 nM, 100 nM. Incubation of cartilageexplants with synovium resulted in increased GAG release. Nanobodyconstruct 581 inhibited GAG release (FIG. 6B).

In line with the GAG release, the analysis of the remaining GAG contentof the cartilage explant (after papain digestion) revealed a decreasedGAG content after co-incubation of explants with synovium compared toexplants alone (FIG. 6C).

Parallel to the analysis of the GAG release, the release of exAGNx1 as aspecific marker for aggrecan degradation was measured. The area underthe curve analysis (AUC) showed a robust induction of exAGNx1 releasewhen cartilage explants and synovium were incubated together. Inaddition, Nanobody construct 581 inhibited exAGNx1 release in aconcentration dependent manner with complete inhibition at 100 nM 581(FIG. 7).

To determine the effect of Nanobody construct 581 on the collagennetwork of the cartilage in the co-culture system, C2M, as a marker forcollagen II degradation was determined. In contrast to the markers fordegradation of the aggrecan network (GAG and exAGNx1), C2M started toincrease between day 14 and 21. Incubation of Nanobody construct 581 inthe co-culture system inhibited release of C2M (FIG. 8).

As additional marker for synovial inflammation, C3M (i.e. a collagentype Ill degradation marker) was determined. A time course analysis ofC3M release indicated that C3M started to increase after 14 days,peaking around day 21. Treatment with Nanobody construct 581 inhibitedC3M in all three concentrations (FIG. 9).

Example 10 Inhibition of Aggrecanase Activity in NHP

The ability of Nanobody construct 581 (2F3^(SO)-35GS linker-Alb11;“C011400581”) to inhibit the aggrecanase activity in vivo was evaluatedin cynomolgus monkey, a non-human primate (NHP) model.

In short, Nanobody construct 581 was given to groups of 3 male and 3female cynomolgus monkeys once weekly by subcutaneous (s.c.)administration at dose levels of 6, 30 or 150 mg/kg for 4 weeks. Aconcomitant control group was treated with the vehicle, i.e. 20 mMHistidine, 8% Sucrose, 0.01% Tween 20 pH 6.0.

Inhibition of the aggrecanase activity was measured on serum samplescollected at several timepoints by determining the level of aggrecandegradation fragments characterized by the presence of the neo-epitopeARGS in serum. All Nanobody construct 581-treated animals showed asimilar profile, i.e. ARGS levels decreased upon first dosing andreached very low levels around or below the lower limit of measuringrange (LLMR) of the assay (0.08 nM) between 48 hours and 120 hours.Subsequently, the ARGS levels showed a sustained maximal decrease aroundor below the LLMR and did not return to baseline by the end of the studyat 4 weeks.

An overview of the results is shown in FIG. 10.

In conclusion, ADAMTS5 specific Nanobodies engage the target followingsystemic administration and potently modulate ARGS neo-epitope levels invivo at all tested dose levels. Moreover, in contrast to the prior artmAbs, no test-item related pathological arrhythmias were observed. Inaddition, there was no evidence for any test-related ST-elevation wasnoted in male and female monkeys treated with the Nanobodies at any ofthe tested dose levels.

Example 11 Inhibition of Cartilage Degeneration

In order to further evaluate the ability of Nanobodies and Nanobodyconstructs to the inhibit cartilage degeneration in vivo, a mouse DMM(destabilization of the medial meniscus) model was used. In short, themedial meniscus was surgically destabilized. Exemplary Nanobodyconstruct 581 (2F3^(SO)-35GS linker-Alb11) was either administeredsubcutaneously 3 days before induction of the DMM (prophylactic) oradministered 3 days after induction of the DMM (therapeutic) at variousconcentrations. Upon 8 weeks of treatment, the animals were sacrificedand knees were removed. The knees were embedded in paraffin blocks,sectioned and stained with toluidine blue. Subsequently, the sectionswere scored for several parameters, including the medial cartilagedegeneration sum.

The results are shown in FIG. 11.

The results show that there is a structural benefit up to 50% for bothprophylactic and therapeutic treatment (s.c.) with Nanobodies in the DMMmouse model.

Example 12 Symptomatic Benefit in Rat Surgical OA Model

In Order to Evaluate the Ability of Nanobodies to Establish aSymptomatic Benefit, a Rat Surgical OA model was used. In short, ratswere treated with ACLt and tMx surgery to induce OA at day 0. In theACLt and tMx surgical model (anterior cruciate ligament transectionextended with a medial meniscectomy) Nanobody construct 581 wasadministered s.c. every other day from day 3 onwards. On a weekly basis,the symptomatic benefit via gait analysis (on a CatWalk) as well asdecrease in joint diameter were determined.

The results are shown in FIG. 12.

Osteoarthritis was induced (at day 0) in adult male rats (Lister Hooded;average body weight of 346±20 g) by anterior cruciate ligamenttransection (ACLT)+resection of the medial meniscus (tMx) surgery at theright knee joint. Treatment with vehicle or test item (sc) started atday 3 after surgery and continued every second day until day 42. Thehealthy control group had no surgical intervention but received scvehicle at the same time points as the dosing groups. (A) Gaitdisturbance over time calculated as “% benefit over vehicle”. Mean ofthe “healthy+placebo” group=100% benefit. Mean of the ““ACLTtMx+vehicle” group=0% benefit. (B) Average “benefit over vehicle” at allinvestigated time points after treatment start. Shown is the mean±SEM of14-15 rats/group. *=p<0.05 calculated with OneWay ANOVA and Dunnett's.

Early treatment with Nanobodies caused a dose-dependent, significant andmeaningful symptomatic benefit during ACLT+tMx induced OA.

Example 13 Toxicity Studies in Cynomolgus Monkeys

A 4-week subchronic toxicity study in cynomogus monkeys was conducted toobtain information on the systemic toxicity, local tolerability andsafety pharmacology of Nanobodies. Exemplary Nanobody construct 581(2F3^(SO)-35GS linker-Alb11) was given s.c. to groups of 3 male and 3female cynomogus monkeys (Macaca fascicularis) (once weekly bysubcutaneous administration into the dorsal region at doses of 6, 30 or150 mg/kg for 4 weeks. A concomitant control group was treated with thevehicle, 20 mM Histidine, 8% Sucrose, 0.01% Tween 20 pH 6.0.

No test item-related signs of local intolerance were noted at any of thetested dose levels. No test item-related effects were noted on thebehavior, the body weight, the food consumption, the hematological andbiochemical parameters, the lymphocytes typing, the CRP levels, theurinalysis parameters, the ophthalmological and auditory functions andthe organ weights of any of the animals at any dose level (data notshown). Neither the macroscopic inspection at necropsy nor thehistopathological examination did reveal any local or systemic organchanges that were related to the treatment with test item in any of theanimals examined at any tested dose level.

Since it has been reported that mAb 12F4 demonstrated focal endocardialhemorrhage, a dose-dependent increase in blood pressure with no evidenceof reversibility as well as cardiac conductance abnormalities (STelevation and ventricular arrhythmias) (Larkin, et al., The highs andlows of translational drug development: Antibody-mediated inhibition ofADAMTS-5 for osteoarthritis disease modification, OARSI conference 2014:Paris; Renninger et al., Identification of Altered CardiovascularFunction Produced by a Novel Biologic Compound in a Stand Alone SafetyPharmacology Primate Study, in SPS meeting, 2013), extensivenon-invasive telemetry employing the EMKA system was performed on theanimals.

No test item-related influence was noted in any of the telemetricparameters (employing the non-invasive EMKA system) of any of theanimals at any dose level. In particular, no pathological arrhythmiaswere observed and there was no evidence for any test item-relatedST-elevation in male and female monkeys treated with the Nanobodies atany of the tested dose levels (data not shown).

These studies confirm that ADAMTS5 specific Nanobodies can be consideredsafe.

Example 14 In Vivo Rat MMT Model DMOAD Study

In order to demonstrate the in vivo efficacy of the ADAMTS5 inhibitorsfused to a CAP binder of the invention, a surgically induced MedialMeniscal Tear (MMT) model in rats was used. In short, an anti-ADAMTS5Nanobody was coupled to a CAP binder (indicated as Nanobody constructC010100954 or Nanobody construct 954). Rats were operated in one knee toinduce OA-like symptoms. Treatment started 3 days post-surgery by IAinjection. Histopathology was performed at day 42 post surgery. Interimand terminal serum samples were taken for exploratory biomarkeranalysis. The medial and total substantial cartilage degeneration widthwere determined, as well as the percentage reduction of cartilagedegeneration. 20 animals were used per group.

The sub-cartilage defect in the medial tibia is shown in FIG. 13.

The results demonstrate that the cartilage width was substantiallyreduced by the ADAMTS5-CAP construct after 42 days compared to thevehicle. These results further suggest that

-   (a) the CAP-moiety has no negative impact on the activity of the    anti-ADAMTS5 Nanobody;-   (b) the CAP-moiety enables the retention of the anti-ADAMTS5    Nanobody; and-   (c) the anti-ADAMTS5 Nanobody has a positive effect on the cartilage    width, even when coupled to a CAP-moiety.

Example 15

Methods: Bovine cartilage explants from four animals (BEX), as well ashuman cartilage explants from eight surgically replaced knee joints(HEX) and from one healthy human knee joint (hHEX), were cultured for upto 21 days in medium alone (w/o), in the presence of pro-inflammatorycytokines (oncostatin M [10 ng/mL]+TNFα [20 ng/mL] (O+T)) or O+T withexemplary Nanobody construct 581 (2F3^(SO)-35GS linker-Alb11) [1 μM-1nM]. Cartilage and synovium from cows (bCC) and from 4 replacedosteoarthritic human knee joints (hCC) were co-cultured ex vivo for upto 28 days in medium alone (w/o), with O+T or O+T plus exemplaryNanobody construct 581 (2F3^(SO)-35GS linker-Alb11) [1 μM-0.6 nM].Cartilage was cut into equal size explants using a biopsy puncher.Synovial membranes were cut into explants of equally sized (30 mg [±3mg]) by scalpel. Metabolic activity of explants was assessed by AlamarBlue. Cartilage tissue turnover was assessed using enzyme-linkedimmunosorbent assay (ELISA) to measure well-characterized biomarkers ofdegradation (huARGS, exAGNxI, C2M) and formation (ProC2) in theconditioned medium. ProC2 and C2M are type II collagen formation anddegradation metabolites, respectively, while exAGNxI and huARGS aremetabolites of ADAMTS-5 degraded aggrecan. Mean values and standarderror of mean (SEM) are reported. Statistical analysis was done usingone-way analysis of variance (ANOVA) with Dunn's multiple comparisonstest or two-way ANOVA with Dunnett's multiple comparisons test assumingnormal distribution.

Results: Metabolic activity of BEX, HEX, and bCC was stable throughoutthe culture period, whereas the metabolic activity in hCC and hHEXdropped significantly from day 14 in O+T treated conditions compared tow/o. In cultures stimulated with O+T, metabolites of ADAMTS-5 degradedaggrecan peaked within the first week of the culture, except for hHEX inwhich huARGS and exAGNxI increased slightly later. Type II collagendegradation, C2M, by O+T peaked after day 19. Type II collagenformation, Pro-C2, remained relatively stable throughout the cultures,compared to the w/o control.

Treatment with exemplary Nanobody construct 581 (2F3^(SO)-35GSlinker-Alb11) in combination with O+T dose dependently decreased huARGSon day 5 in BEX (highest dose: 8% of O+T), HEX (highest dose: 40% ofO+T), bCC (highest dose: 10% of O+T), hCC (highest dose: 40% of O+T),and hHEX (highest dose: 24% of O+T) (FIG. 14). The IC50 of exemplaryNanobody construct 581 (2F3^(SO)-35GS linker-Alb11) based on thereduction of huARGS ranged from 300 nM in BEX to <15 nM HEX, hHEX, bCCand hCC (FIG. 14). The effect of exemplary Nanobody construct 581(2F3^(SO)-35GS linker-Alb11) on exAGNxI was similar to huARGS in thecultures tested. Exemplary Nanobody construct 581 (2F35^(SO)-35GSlinker-Alb11) also reduced C2M (marker for type II collagen degradation)significantly and dose dependently, albeit the effect was less than foraggrecan degradation markers. exemplary Nanobody construct 581(2F3^(SO)-35GS linker-Alb11) showed no effect on type II collagenformation metabolite Pro-C2 in any of the tested conditions.

Conclusions: Here, we have shown that the exemplary Nanobody construct581 (2F3^(SO)-35GS linker-Alb11) has cartilage protective effects due toits dose-dependent inhibition of ADAMTS-5-mediated aggrecan degradationand MMP-mediated type II collagen degradation in pro-inflammatoryconditions of bovine and human cartilage ex vivo cultures and inco-cultures of cartilage and synovium. 2F3^(SO)-35GS linker-Alb (SEQ IDNO: 129) is one of the preferred embodiments of the invention.

Example 16 Present Nanobody Constructs Outperform Prior Art Nanobodies

In WO2008/074840 various ADAMTS5 Nanobodies have been described. In thepresent experiment a comparison was made between the 26 Nanobodiesdescribed in WO2008/074840 versus the Nanobodies of the presentinvention represented by the exemplary Nanobody 2F3^(SO) (2F3*).

All constructs were tested in the AlphaLSA assay, as described inExample 5.1. Nanobody 2F3^(SO) (2F3*) is the ADAMTS5 binding monovalentbuilding block of C011400581 (SEQ ID NO: 129). As a negative control,the irrelevant Nanobody IRR00028 was used.

First the activity of the prior art Nanobodies was determined in theAlphaLSA. The results are shown in Table 16.1.

TABLE 16.1 Activity of the prior art Nanobodies in Alpha LISA CDR3AlphaLisa No Name Length length Results 1 36A01 149 17 Blocking 2 36A06147 15 enhancer 3 36C06 150 17 Enhancer 4 36D06 146 14 Blocking 5 36E01147 15 Enhancer 6 36F01 149 17 Blocking 7 37B01 146 15 Enhacer 8 37B06145 13 Blocking 9 37B12 151 16 Enhancer 10 37C06 150 18 Enhancer 1137C12 150 18 Enhancer 12 37D06 146 15 Enhancer 13 37E06 151 16 NoActivity 14 37F01 146 15 Enhancer 15 37F12 150 18 Enhancer 16 37G01 15018 Blocking 17 37G06 146 15 Enhancer 18 40A07 151 16 No Activity 1940B08 146 15 No Activity 20 40D07 146 14 No Activity 21 40E08 147 15 NoActivity 22 40F07 146 14 Blocking 23 40F08 151 19 No Activity 24 40G07146 14 Blocking 25 40G08 151 16 No Activity 26 40H07 146 14 No Activity

The results demonstrate that only 7 prior art Nanobodies have a blockingactivity. The remainder of the prior art Nanobodies had no activity orenhanced the activity.

In a second phase of this comparative experiment, the blockingNanobodies were compared vis-à-vis the exemplary monovalent Nanobody2F3^(so) (2F3*).

The IC₅₀ values of the Nanobody constructs are listed in Table 16.2. Thefold-difference with the exemplary monovalent Nanobody 2F3^(so) (2F3*)is also indicated for ease of comparison.

TABLE 16.2 Potencies of prior art Nanobody constructs vis-à-vis Nanobody2F3^(SO) (2F3*) as determined in AlphaLISA Fold- difference vs Exp ID NbID IC50 [M] 2F3^(SO) (2F3*) 1 2F3^(SO) (2F3*) 2.0E−10 [1.2E−10; 3.1E−10]— 36D06 1.4E−09 [8.5E−10; 3.6E−09]  7× 36F01 7.0E−09 [2.3E−09] 35× 36B062.2E−09 [1.4E−09; 3.7E−09] 11× 2 2F3^(SO) (2F3*) 2.7E−10 [2.2E−10;3.4E−10] — 37G01 7.3E−09 [5.2E−09; 1.2E−08] 27× 3 2F3^(SO) (2F3*)1.9E−10 [1.5E−10; 2.3E−10] — 40F07 1.9E−09 [1.4E−09; 2.8E−09] 10× 40G071.7E−09 [1.2E−09; 2.4E−09]  9× 4 2F3^(SO) (2F3*) 4.4E−10 [3.6E−10;5.3E−10] C011400581 3.5E−10 [2.8E−10; 4.4E−10] 36A01 Partial inhibition

It can be concluded that the Nanobody constructs of the presentinvention outperformed the prior art ADAMTS5 Nanobodies.

TABLE A-1Name and short description (“ID”), SEQ ID NO:s (“SEQ”) and amino acid sequences ofmonovalent and multivalent anti-ADAMTS5 Nanobodies ID SEQ Sequence 2A025EVQLVESGGGLVQPGGSLRLSCAASRRTFSSYVMAWFRQAPGKEREFVAAISRSGDSTYYYDSLEGRFTISRDNAKNTVHLQMNSLKPEDTAVYICAASRAPSFRTIDAINYYDYWGQGTLVTVSS 2A12 1EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERDFVAGISRSAGRTYYVDSVKGRFTISRDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSS 2C10 9EVQLVESGGGLVQAGGSLRLSCATSGFTFSPYYMGWFRQAPGKERDFVAAITRSRGTTYYLDSTEGRFTISRDNAKNTMYLQMNSLNPEDTAVYYCAAGRSPGDPSRTYLYEYWGQGTLVTVSS 2D07 6EVQLVESGGGLVQAGGSLRLSCSFSGPGRTFARYAMGWFRQAPGKNRDFITGISGSGDSTYYVYPMKDRFTISRDNAKNMVYLQMNALKPEDTAVYYCAADREINRIANDKELDFWGQGTLVTVSS 2D12 4EVQLVESGGGLVQAGDSLRLSCAASGRTFSTYFVGWFRQAPGKERDFVAAISRNGARTYYYDSVAGLFTISRDNAKNTVYLQMSSLKPEDTAVYYCAAARISPSDPSNEDGYDYWGQGTLVTVSS 2F03 2EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTISRDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSS 2G01 7EVQLVESGGGLVQAGGSLRLSCAASGRTTFSSYAMGWFRQAPGKERAFVATIWSGGLTVYADSAKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAAEAVGTYYTPDGWTYWGQGTLVTVSS 3B02 16EVQLVESGGGFVQAGGSLRLSCVASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGMPYYLDSVMDRFTISRDNAKNTVSLQMNSLQPEDTAVYYCAADRSAFRDPSFDVNYEYWGRGTLVTVSS 3B03 13EVQLVESGGDLVQPGGSLRLSCAASGSDVVVNDMGWYRQAPGKQRELVADITTGGRTNYADSVKGRFTISRDNVKNTVYLQMNSLKPEDTAVYYCNAQVGDSDDDVWYAYWGQGTLVTVSS 3D01 10EVQLVESGGGLVQAGGSLRLSCAPSGFTFSPYYMGWFRQAPGKERDFVAAISRSRGTTYYLDSTEGRFTISRDNANDTVYLQMNSLNPEDTAVYYCAAGRSPGDPSRTYLYDYWGQGTLVTVSS 3D02 11EVQLVESGGGLVQAGASLRLSCATSGFTFSPYYMGWFRQAPGKERDFVAAISWSRGILYYTDSTEGRFTISRDNAKNTMYLQMDNLNPEDTAVYYCAASRSPGDPSRTYLYDYWGQGTLVTVSS 7B11 14EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAIISGGRTNYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCNAEVDAGIYAYGYWGQGTLVTVSS 9A05 17EVQLVESGGGFVQAGGSLRLSCAASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGITFYPDSVEGRFTISGDNAKNTVSLQMNSLKPEDTAVYYCAADRSALRDPSFEVNYEYWGRGTLVTVSS 9D03 3EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIARDSAKNTVYLQMNPLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSS 9D09 15EVQLVESGGGLVQAGGSLRLSCAASGLTFSSYTMGWFRQAPGQEREFVSAISWNTFTTYYVDSVKDRFTVSRDNAKNTLYLRMNSLKPEDTAVYYCAAAGGSPRQHEPYEYRVWGQGTLVTVSS 9D10 12EVQLVESGGGLVQAGGSLRLSCAASGRALSSSIMGWFRQAPGKEREFVAAITWSGGRAYYADVSDFEKGRFTISRDNGKNTVNLQMKGLKPEDTAVYYCAAALAIPVTMSPHEYPYWGQGTLVTVSS 11B06 8EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGINWSGGTTYYVDSVKGRFTISRDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSS 13E02 18EVQLVESGGGLVQAGGSLRLSCVASGSIFSIDAMGWYRQAPGKERELVASVTTGASPNYGDSVTGRFTASRDRAKNALYLQMNSLKPEDTAVYYCNLIMTIPGGSQIMYWGQGTLVTVSS 3F04 19EVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDNYSDWGQGTLVTVSS construct 117EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTIS049 RDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSS 11B06: N52construct 116EVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYA093 KNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSS 3F04: N100 fQconstruct 120EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERDFVAGISRSAGRTYYVDSVKGRFTIS004 2A12-RDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGAlbGSGGGGSGGGGSGGGGSEVQLVESCGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct121EVQLVESGGGLVQAGGSLRLSCSFSGPGRTFARYAMGWFRQAPGKNRDFITGISGSGDSTYYVYPMKDRFT005 2D7-ISRDNAKNMVYLQMNALKPEDTAVYYCAADREINRIANDKELDFWGQGTLVTVSSGGGGSGGGGSGGGGSGAlbGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSconstruct 122EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTIS006 2F3-RDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGAlbGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct123EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTIS069 049-RDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGAlbGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct124EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIA070 9D3-RDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGAlbGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGETFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct125EVQLVESGGGFVQAGGSLRLSCVASRRTISSGTMGWFRQAPGKEREFVAAIRWSSGMPYYLDSVMDRFTIS071 3B2 -RDNAYNTVSLQMNSLQPEDTAVYYCAADRSAFRDPSFDVNYEYWGRGTLVTVSSGGGGSGGGGSGGGGSGGAlbGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct126EVQLVESGGGLVQAGGSLRLSCAASGRTVSSYAMGWFRLAPGKEREFVAGISRSAERTYYVDSLKGRFTIS129 2F3 -RDSAKNTVYLHMNRLKPEDTAVYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGG093-AlbGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREEVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct 127EVQLVESGGGLVQAGGSLRLSCAASGLTFRRNAMGWFRQAPGKERELLAGISWSGGTTYYVDSVKGRFTIS130 049-RDNAKNTVDLQMISPKPEDTAVYYCAADGDIGTLVNDENPRYWGQGTLVTVSSGGGGSGGGGSGGGGSGGG093-AlbGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct 128EVQLVESGGGLVQSGGSLRLSCAASGSAVSVNAMAWYRQAPGKQRDFVAGISRSAGRTYYTDSVKDRFTIA131 9D3-RDSAKNTVYLQMNRLKPEDTAVYYCAADLDPNRIFSRDEAAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGG093 -AlbGSGGGGSGGGGSGGGGSEVQLVESGGGSVQAGGSLRLSCVASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVQGRFTVSRDYAKNTLYLQMNSLKPEDAAVYYCAGDPWGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS construct 129DVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTIS577 = 581RDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGG2F3*-AlbGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYYCTIGGSLSRSSQGTLVTVSSAconstruct 130DVQLVESGGGVVQPGGSLRLSCAASGRTVSSYAMGWFRQAPGKEREFVAGISRSAERTYYVDSLKGRFTIS579 2F3*-RDNSKNTVYLQMNSLRPEDTALYYCAADLDPNRIFSREEYAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGG093*-AlbGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRYPMAWFRQAPGKEREFVAGVSWGGDRTYYADSVKGRFTISRDYSKNTLYLQMNSLRPEDTALYYCAGDPFGRLFRVKDQYSDWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA *SO (sequence optimized) version

TABLE A-2Sequences for CDRs and frameworks, plus preferred combinations as provided in formula I, namelyFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (the following terms: “ID” refers to the given SEQ ID NO)ID Nanobody ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 1 02A12 72 EVQLVESGGGLV20 GRTFSSYAMG 85 WFRQAPGK 36 GISRSAGRTY 95 YVDSVKGRFTISRDSAKNTVAGGSLRLSCAAS ERDFVA YLQMNRLKPEDTAVYYCAA 2 02F03 72 EVQLVESGGGLVQ 21GRTVSSYAMG 86 WFRLAPGK 37 GISRSAERTY 96 YVDSLKGRFTISRDSAKNTVAGGSLRLSCAAS EREFVA YLHMNRLKPEDTAVYYCAA 3 09D03 73 EVQLVESGGGLVQ 22GSAVSVNAMA 87 WYRQAPGK 36 GISRSAGRTY 97 YTDSVKDRFTIARDSAKNTVSGGSLRLSCAAS QRDEVA YLQMNRLKPEDTAVYYCAA 4 02D12 74 EVQLVESGGGLVQ 23GRTFSTYFVG 85 WERQAPCK 38 AISRNGARTY 98 YDSVAGLFTISRDNAKNTVYAGDSLRLSCAAS ERDFVA Y LQMSSLKPEDTAVYYCAA 5 02A02 75 EVQLVESGGGLVQ 24RRTFSSYVMA 88 WERQAPGK 39 AISRSGDSTY 99 YYDSLEGRFTISRDNAKNTVPGGSLRLSCAAS EREFVA HLQMNSLKPEDTAVYICAA 6 02D07 76 EVQLVESGGGLV 25GPGRTFARYAM 89 WERQAPGK 40 GISGSGDSTY 100 YVYPMKDRFTISRDNAKNMVAGGSLRLSCSFS G NRDFIT YLQMNALKPEDTAVYYCAA 7 02G01 72 EVQLVESGGGLVQ 26GRTTFSSYAMG 90 WERQAPGK 41 TIWSGGLTV 101 YADSAKGRFTISRDNAKNTVAGGSLRLSCAAS ERAFVA YLQMNSLRPEDTAVYYCAA 8 11B06 72 EVQLVESGGGLVQ 27CLTFRRNAMG 91 4ERQAPGK 42 GINWSGGTTY 102 YVDSVKGRFTISRDNAKNTVAGGSLRLSCAAS ERELLA DLQMISPKPEDTAVYYCAA 117 49 72 EVQLVESGGGLVQ 27GLTFRRNAMG 91 WFRQAPGK 119 GISWSGGTTY 102 YVDSVKGRFTISRDNAKNTV 11B06:AGGSLRLSCAAS ERELLA DLQMISPKPEDTAVYYCAA N52S 9 02C10 77 EVQLVESGGGLVQ 28GFTFSPYYMG 85 WERQAPGK 43 AITRSRGTTY 103 YLDSTEGRFTISRDNAKNTMAGGSLRLSCATS ERDFVA YLQMNSLNPEDTAVYYCAA 10 03D01 78 EVQLVESGGGLV 28GFTFSPYYMG 85 WERQAPGK 44 AISRSRGTTY 104 YLDSTEGRFTISRDNANDTVAGGSLRLSCAPS ERDFVA YLQMNSLNPEDTAVYYCAA 11 03D02 79 EVQLVESGGGLVQ 28GFTFSPYYMG 85 WERQAPGK 45 AISWSRGILY 105 YTDSTEGRFTISRDNAKNTMAGASLRLSCATS ERDFVA YLQMDNLNPEDTAVYYCAA 12 09D10 72 EVQLVESGGGLVQ 29GRALSSSIMG 88 WERQAPGK 46 AITWSGGRAY 106 VSDFEKGRFTISRDNGKNTVAGGSLRLSCAAS EREFVA YAD NLQMKGLKPEDTAVYYCAA 13 03B03 80 EVQLVESGGDLVQ 30GSDVVVNDMG 92 WYRQAPGK 47 DITTGGRTN 107 YADSVKGRFTISRDNVKNTVPGGSLRLSCAAS QRELVA YLQMNSLKPEDTAVYYCNA 14 07B11 75 EVQLVESGGGLVQ 31GSIFSINVMG 92 WYRQAPGK 48 AIISGGRTN 108 YADSVKGRFTISRDNSKNTVPGGSLRLSCAAS QRELVA YLQMNSLRPEDTAVYYCNA 15 09D09 72 EVQLVESGGGLVQ 32GLTFSSYTMG 93 WERQAPGQ 49 AISWNTETTY 109 YVDSVKDRFTVSRDNAKNTLAGGSLRLSCAAS EREFVS YLRMNSLKPEDTAVYYCAA 16 03B02 81 EVQLVESGGGFVQ 33RRTISSGTMG 88 WERQAPGK 50 AIRWSSGMPY 110 YLDSVMDRFTISRDNAKNTVAGGSLRLSCVAS EREFVA SLQMNSLQPEDTAVYYCAA 17 09A05 82 EVQLVESGGGFVQ 33RRTISSGTMG 88 WERQAPCK 51 AIRWSSGITF 111 YPDSVEGRFTISGDNAKNTVAGGSLRLSCAAS EREFVA SLQMNSLKPEDTAVYYCAA 18 13E02 83 EVQLVESGGGLVQ 34GSIFSIDAMG 94 WYRQAPGK 52 SVTTGASPN 112 YGDSVTGRFTASRDRAKNALAGGSLRLSCVAS ERELVA YLQMNSLKPEDTAVYYCNL 19 03F04 84 EVQLVESGGGSVQ 35GRYPMA 88 WFRQAPGK 53 GVSSIGGDRTY 113 YADSVQGRFTVSRDYAKNTL AGGSLRLSCVASEREFVA YLQMNSLKPEDAAVYYCAG 116 93(3F04 84 EVQLVESGGGSVQ 35 GRYPMA 88WFRQAPGK 53 GVSWGGDRTY 113 YADSVQGRFTVSRDYAKNTL N100fQ) AGGSLRLSCVASEREFVA YLQMNSLKPEDAAVYYCAG ID Nanobody ID CDR3 ID FR4 1 02A12 54DLDPNRIFSRDEAA 114 WGQGTL Y VTVSS 2 02F03 55 DLDPNRIFSREEYA 114 WGQGTL YVTVSS 3 09D03 54 DLDPNRIFSRDEAA 114 WGQGTL Y VTVSS 4 02D12 56ARISPSDPSNEDGY 114 WGQGTL DY VTVSS 5 02A02 57 SRAPSFRTIDAINY 114 WGQGTLYDY VTVSS 6 02D07 58 DREINRIANDKELD 114 WGQGTL F VTVSS 7 02G01 59EAVGTYYTPDGWTY 114 WGQGTL VTVSS 8 11B06 60 DGDIGTLVNDENPR 114 WGQGTL YVTVSS 117 49 60 DGDIGTLVNDENPR 114 WGQGTL 11B06: Y VTVSS N52S 9 02C10 61GRSPGDPSRTYLYE 114 WGQGTL Y VTVSS 10 03D01 62 GRSPGDPSRTYLYD 114 WGQGTLY VTVSS 11 03D02 63 SRSPGDPSRTYLYD 114 WGQGTL Y VTVSS 12 09D10 64ALAIPVTMSPHEYP 114 WGQGTL Y VTVSS 13 03B03 65 QVGDSDDDVWYAY 114 WGQGTLVTVSS 14 07B11 66 EVDAGIYAYGY 114 WGQGTL VTVSS 15 09D09 67AGGSPRQHEPYEYR 114 WGQGTL V VTVSS 16 03B02 68 DRSAFRDPSEDVNY 115 WGRGTLEY VTVSS 17 09A05 69 DRSALRDPSFEVNY 115 WGQGTL EY VTVSS 18 13E02 70IMTIPGGSQIMY 114 WGQGTL VTVSS 19 03F04 71 DPWGRLFRVKDNYS 114 WGQGTL DVTVSS 116 93(3F04 118  DPWGRLFRVKDQYS 114 WGQGTL N100fQ) D VTVSS

TABLE BMiscellaneous Amino acid sequences: Name and short description (“ID”), SEQ ID NO:s (“SEQ”)and amino acid sequences (“sequences”) SEQ species IDAmino acid sequence human 149MLLGWASLLLCAFRLPLAAVGPAATPAQDKAGQPPTAAAAAQPRRRQGEEVQERAEPPGHPHPLAQRRRSKGLVQNIADAM-TS5DQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGIAGFVRAGGGTSAPWRHRSHCFYRGTVDGSPRSLAVFDLCGGLDGQ9UNA0-1FFAVKHARYTLKPLLRGPWAEEEKGRVYGDGSARILHVYTREGFSFEALPPRASCETPASTPEAHEHAPAHSNPSGRAALASQLLDQSALSPAGGSGPQTWWARRRRSISRARQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFKYRHCNNPAPRNNGKYCTGKRAIYRSCSLMPCPPNGKSFRHEQCEAKNGYQSDAKGVKTFVEWVPKYAGVLPADVCKLTCRAKGTGYYVVFSPKVTDGTECRLYSNSVCVRGKCVRTGCDGIIGSKLQYDKCGVCGGDNSSCTKIVGTFNKKSKGYTDVVRIPEGATHIKVRQFKAKDQTRFTAYLALKKKNGSYLINGKYMISTSETIIDINGTVMNYSGWSHRDDFLHGMGYSATKEILIVQILATDPTKPLDVRYSFFVPKKSTPKVNSVTSHGSNKVGSHTSQPQWVTGPWLACSRTCDTGWHTRTVQCQDGNRKLAKGCPLSQRPSAFKQCLLKKC bovine 150MLLGWAALMLCALRLPPVAAGPTAAPAQDKAGQPRAAAVAAAAQPRGARGEEAQEPAEPPGHPHPLAPQRGSRGLVQ(AANIDQLYSGGGKVGYLVYAGGRRFLLDLERDDSVGAAGLVPAGGGPNATRRHRGHCFYRGTVDGSPRSLAVFDLCGGLresiduesDGFFAVKRARYTLQPLLRGPWAEAEGDARVYGDESARILHVYTREGFSFEALPPRTSCETHASPPGARERPPAPSRP1-626)DGRWALAPQQLPGQSAPSSDGSQGPRTWWRRPRRSISRARQVELLLVADASMARMYGRGLQHYLLTLASIANKLYSHASIENHIRLVVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQIPGPEELPGQTYDASQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPANNGRYCTGKRAIYRSCSVTPCPHHHHHHHHHH rat 151MRLEWASLLLLLLLLCASCLALAADNPAAAPAQDKTRQPRAAAAAAQPDQRQWEETQERGHPQPLARQRRSSGLVQN(AAIDQLYSGGGKVGYLVYAGGRRFLLDLERDDTVGAAGGIVTAGGLSASSGHRGHCFYRGTVDGSPRSLAVFDLCGGLDresiduesGPFAVKHARYTLKPLLRGSWAESERVYGDGSSRILHVYTREGFSFEALPPRTSCETPASPSGAQESPSVHSSSRRRT1-619)ELAPQLLDHSAFSPAGNAGPQTWWRRRRRSISRARQVELLLVADSSMAKMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLTDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDVPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGRYCTGKRAIYRSCSVIPCPHHHHHHHHHH guinea pig 152MLLGWASLLLCAFRLPQAAASAAAAPAQDKAGQPRAAAAAPQPRRRQGEHAPLRVEPPGHPHALAPQRRGRGLLQSI(AADRLYSGGGKVGYLVYAGGRRFLLDLERDGSVGAAGLFPAGGGLSAPRRNRSHCFYRGTVDGSPRSLAVFDLCGGLRGresiduesFFAVKHARYTVKPLLRGPWAEADTPRVYGDESARIPHVYTREGFSFEALPPRASCETPASQPGPHERPPAHNSPGRH1-622)STVDPQLPELSALSPAGDPGQQIWWRRRRRSISRARQVELLLVADGSMAKMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGLTEDKRLMSSILTSIDASKPWSKCTSATMTEFLDDGHGNCLLDVPRKQIPSPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGRYCTGKRAIYRSCSVTPCPHHHHHHHHHH mouse 153MRLEWAPLLLLLLLLSASCLSLAADSPAAAPAQDKTRQPQAAAAAAEPDQPQGEETRERGHLQPLAGQRRSGGLVQN(AAIDQLYSGGGKVGYLVYAGGRRFLLDLERDDTVGAAGSIVTAGGGLSASSGHRGHCKYRGTVDGSPRSLAVFDLCGGLresiduesDGFFAVKHARYTLKPLLRGSWAEYERIYGDGSSRILHVYNREGFSFEALPPRASCETPASPSGPQESPSVHSRSRRR1-622)SALAPQLLDHSAFSPSGNAGPQTWWRRRRRSISRARQVELLLVADSSMARMYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLTDKDTSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEENFGTTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRVCLQGKCVDKTKKKYYSTSSHGNWGSWGPWGQCSRSCGGGVQFAYRHCNNPAPRNSGWYCTGKRAIYRSCSVTPCPHHHHHHHHHH cynomolgus 154MLLGWASLLLCAFRLPLAAAGPAAAPAQDKAGQPATAAAAAQPRRRQGEEVQERTEPPGHPHPLAQRRSSKGLVQNImonkeyDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGTAGFVPTEGGTSAPWRHRSHCFYRGTVDGSPRSLAVEDLCGGLDG(AAFFAVKHARYTLKPLLRGPWAEEETRRVYGDGSARILHVYTREGFSFEALQPRASCETPASTTEPHERPPAHSNPGGRresiduesAALASQLLDQSAVSPAGGPGPQTWWRRRRRSISRAPQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIE1-622)NHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDQPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCDTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCGLMPCPHHHHHHHHHH human 155MTTLLWVFVTLRVITAAVTVETSDHDNSLSVSIPQPSPLRVLLGTSLTIPCYFIDPMHPVTTAPSTAPLAPRIKWSRaggrecanVSKEKEVVLLVATEGRVRVNSAYQDKVSLPNYPAIPSDATLEVQSLRSNDSGVYRCEVMHGIEDSEATLEVVVKGIVFHYRAISTRYTLDFDRAQRACLQNSAIIATPEQLQAAYEDGFHQCDAGWLADQTVRYPIHTPREGCYGDKDEFPGVRTYGIRDTNETYDVYCFAEEMEGEVFYATSPEKFTFQEAANECRRLGARLATTGHVYLAWQAGMDMCSAGWLADRSVRYPISKARPNCGGNLLGVRTVYVHANQTGYPDPSSRYDAICYTGEDFVDIPENFFGVGGEEDITVQTVTWPDMELPLPRNITEGEARGSVILTVKPIFEVSPSPLEPEEPFTFAPEIGATAFAEVENETGEATRPWGFPTPGLGPATAFTSEDLVVQVTAVPGQPHLPGGVVFHYRPGPTRYSLTFEEAQQACPGTGAVIASPEQLQAAYEAGYEQCDAGWLRDQTVRYPIVSPRTPCVGDKDSSPGVRTYGVRPSTETYDVYCFVDRLEGEVFFATRLEQFTFQEALEFCESHNATATTGQLYAAWSRGLDKCYAGWLADGSLRYPIVTPRPACGGDKPGVRTVYLYPNQTGLPDPLSRHHAFCFRGISAVPSPGEEEGGTPTSPSGVEEWIVTQVVPGVAAVPVEEETTAVPSGETTAILEFTTEPENQTEWEPAYTPVGTSPLPGILPTWPPTGAETEESTEGPSATEVPSASEEPSPSEVPFPSEEPSPSEEPFPSVRPFPSVELFPSEEPFPSKEPSPSEEPSASEEPYTPSPPEPSWTELPSSGEESGAPDVSGDFTGSGDVSGHLDFSGQLSGDRASGLPSGDLDSSGLTSTVGSGLTVESGLPSGDEERIEWPSTPTVGELPSGAEILEGSASGVGDLSGLPSGEVLETSASGVGDLSGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETTAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETAAPGVEDISGLPSGEVLETTAPGVEEISGLPSGEVLETTAPGVDEISGLPSGEVLETTAPGVEEISGLPSGEVLETSTSAVGDLSGLPSGGEVLEISVSGVEDISGLPSGEVVETSASGIEDVSELPSGEGLETSASGVEDLSRLPSGEEVLEISASGFGDLSGVPSGGEGLETSASEVGTDLSGLPSGREGLETSASGAEDLSGLPSGKEDLVGSASGDLDLGKLPSGTLGSGQAPETSGLPSGESGEYSGVDLGSGPTSGLPDFSGLPSGFPTVSLVDSTLVEVVTASTASELEGRGTIGISGAGEISGLPSSELDISGRASGLPSGTELSGQASGSPDVSGEIPGLFGVSGQPSGFPDTSGETSGVTELSGLSSGQPGVSGEASGVLYGTSQPFGITDLSGETSGVPDLSGQPSGLPGFSGATSGVPDLVSGTTSGSGESSGITFVDTSLVEVAPTTEKEEEGLGSVELSGLPSGEADLSGKSGMVDVSGQFSGTVDSSGFTSQTPEFSGLPSGIAEVSGESSRAEIGSSLPSGAYYGSGTPSSEPTVSLVDRTLVESVTQAPTAQEAGEGPSGILELSGAHSGAPDMSGEHSGFLDLSGLQSGLIEPSGEPPGTPYFSGDFASTTNVSGESSVAMGTSGEASGLPEVTLITSEFVEGVTEPTISQELGQRPPVTHTPQLFESSGKVSTAGDISGATPVLPGSGVEVSSVPESSSETSAYPEAGFGASAAPEASREDSGSPDLSETTSAFHEANLERSSGLGVSGSTLTFQEGEASAAPEVSGESTTTSDVGTEAPGLPSATPTASGDRTEISGDLSGHTSQLGVVISTSIPESEWTQQTQRPAETHLEIESSSLLYSGEETHTVETATSPTDASIPASPEWKRESESTAAAPARSCAEEPCGAGTCKETEGHVICLCPPGYTGEHCNIDQEVCEEGWNKYQGHCYRHFPDRETWVDAERRCREQQSHLSSIVTPEEQEFVNNNAQDYQWIGLNDRTIEGDFRWSDGHPMQFENWRPNQPDNFFAAGEDCVVMIWHEKGEWNDVPCNYHLPFTCKKGTVACGEPPVVEHARTFGQKKDRYEINSLVRYQCTEGFVQRHMPTIRCQPSGHWEEPRITCTDATTYKRRLQICRSSRHPRRSRPSTAR 00745 156EVQLVESGGGVVQPGGSLRLSCAASGSTFIINVVRWYRRAPGKQRELVATISSGGNANYVDSVRGRFTISRDNSKNTPEA114F08 VYLQMNSLRPEDTALYYCNVPTTHYGGVYYGPYWGQGTLVTVSSA 00747 157EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYTMGWFRQAPGKEREFVAAISWSGGRTYYADSVKGRFTISRDNSKNPEA604F02 TVYLQMNSLRPEDTALYYCAAYRRRRASSNRGLWDYWGQGTLVTVSSA

TABLE CVarious Linker sequences (“ID” refers to the SEQ ID NO as used herein)Name ID Amino acid sequence A3 158 AAA 5GS linker 159 GGGGS 7GS linker160 SGGSGGS 8GS linker 161 GGGGGGGS 9GS linker 162 GGGGSGGGS 10GS linker163 GGGGSGGGGS 15GS linker 164 GGGGSGGGGSGGGGS 18GS linker 165GGGGSGGGGSGGGGGGGS 20GS linker 166 GGGGSGGGGSGGGGSGGGGS 25GS linker 167GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 168 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS35GS linker 169 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS linker 170GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 171 EPKSCDKTHTCPPCP9GS-G1 hinge 172 GGGGSGGGSEPKSCDKTHTCPPCP Llama upper long 173EPKTPKPQPAAA hinge region G3 hinge 174ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPP PCPRCPEPKSCDTPPPCPRCP

TABLE DSerum albumin binding ISVD sequences (“ID” refers to the SEQ ID NO as used herein),including the CDR sequences Name ID Amino acid sequence Alb8 131EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 132EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 133EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 134EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 135EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11 136EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADS(S112K)-A VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82137 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 138EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 139EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 140EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 141EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GG 142EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 143EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb92 144EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 145EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA ALB CDR1 146 SFGMSALB CDR2 147 SISGSGSDTLYADSVKG ALB CDR3 148 GGSLSR

1. A polypeptide comprising at least 1 immunoglobulin single variabledomain (ISVD) binding an A Disintegrin and Metalloproteinase withThrombospondin motifs (ADAMTS). 2.-3. (canceled)
 4. The polypeptideaccording to claim 1, wherein said ISVD binding ADAMTS5 comprises 3complementarity determining regions, wherein the complementarydetermining regions are CDR1 to CDR3, in which (i) CDR1 is selected fromthe group consisting of SEQ ID NOs: 21, 35, 20, 22, 25, 33, 28, 24, 23,26, 27, 29, 30, 31, 32 and 34; and amino acid sequences that have 1, 2or 3 amino acid difference(s) with SEQ ID NOs: 21, 35, 20, 22, 25, 33,33, 28, 24, 23, 26, 27, 29, 30, 31, 32 and 34; (ii) CDR2 is selectedfrom the group consisting of SEQ ID NOs: 37, 53, 36, 40, 50, 51, 44, 45,43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52; and amino acid sequencesthat have 1, 2 or 3 amino acid difference(s) with SEQ ID NOs: 37, 53,36, 40, 50, 51, 44, 45, 43, 39, 38, 41, 119, 42, 46, 47, 48, 49 and 52;and (iii) CDR3 is selected from the group consisting of SEQ ID NO: SEQID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63, 61, 57, 56, 59, 60, 64, 65,66, 67 and 70; and amino acid sequences that have 1, 2, 3 or 4 aminoacid difference(s) with SEQ ID NOs: 55, 118, 71, 54, 58, 68, 69, 62, 63,61, 57, 56, 59, 60, 64, 65, 66, 67 and
 70. 5.-10. (canceled)
 11. Thepolypeptide according to claim 1, wherein said polypeptide is SEQ ID NO:129 or 130 or a polypeptide which has at least 95% sequence identity toSEQ ID NO: 129 or
 130. 12. (canceled)
 13. The polypeptide according toclaim 4, in which said ISVD is chosen from the group consisting of SEQID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8, 117, 12, 13,14, 15 and
 18. 14.-21. (canceled)
 22. The polypeptide according to claim1, wherein said polypeptide inhibits the protease activity of ADAMTS5.23. The polypeptide according to claim 1, comprising at least 2 ISVDs,wherein at least 1 ISVD specifically binds ADAMTS and is selected fromthe group consisting of SEQ ID NO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11,9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and
 18. 24. (canceled)
 25. Apolypeptide comprising two or more ISVDs which each individuallyspecifically bind ADAMTS5, wherein a) at least a first ISVD specificallybinds a first antigenic determinant, epitope, part, domain, subunit orconformation of ADAMTS5; and wherein, b) at least a second ISVDspecifically binds a second antigenic determinant, epitope, part,domain, subunit or conformation of ADAMTS5, different from the firstantigenic determinant epitope, part, domain, subunit or conformation,respectively.
 26. The polypeptide according to claim 25, wherein saidfirst ISVD specifically binding ADAMTS5 is selected from the groupconsisting of SEQ ID NO:s 2, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 8,117, 12, 13, 14, 15 and
 18. 27. The polypeptide according to claim 25,wherein said second ISVD specifically binding ADAMTS5 is SEQ ID NO: 118or
 19. 28. The polypeptide according to claim 25, wherein thepolypeptide comprises a polypeptide chosen from the group consisting ofSEQ ID NO: 126 (clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130049-093-Alb) and SEQ ID NO: 128 (clone 131 9D3-093-Alb) or a polypeptidethat has an amino acid sequence identity of at least 95% to SEQ ID NO:126, 127, 128, 130 or
 131. 29. The polypeptide according to claim 1,further comprising an ISVD binding serum albumin. 30.-31. (canceled) 32.The polypeptide according to claim 29, wherein the polypeptide isselected from the group consisting of SEQ ID NO: 129 (clone 5772F3^(SO)-Alb), SEQ ID NO: 130 (clone 579 2F3^(SO)-093-Alb), SEQ ID NO:120 (clone 4 2A12-Alb), SEQ ID NO: 121 (clone 5 2D7-Alb), SEQ ID NO: 122(clone 6 2F3-Alb), SEQ ID NO: 123 (clone 69 049-Alb), SEQ ID NO: 124(clone 70 9D3-Alb), SEQ ID NO: 125 (clone 71 3B2-Alb), SEQ ID NO: 126(clone 129 2F3-093-Alb), SEQ ID NO: 127 (clone 130 049-093-Alb), and SEQID NO: 128 (clone 131 9D3-093-Alb).
 33. The polypeptide according toclaim 1 further comprising at least one ISVD specifically bindingAggrecan. 34.-44. (canceled)
 45. The polypeptide according to claim 1,wherein said polypeptide has at least 80%, 90%, 95% or 100% sequenceidentity with any of SEQ ID NO:s 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 116, 117, 120, 121, 122, 123, 124, 125, 126,127, 128, 129 or
 130. 46. A method of treating and/or preventing adisease or disorder in an individual, comprising administering thepolypeptide according to claim 1 to said individual in an amounteffective to treat or prevent said disease or disorder.
 47. The methodaccording to claim 46, wherein said disease or disorder is selected fromthe group consisting of arthropathies, chondrodystrophies, arthriticdisease, osteoarthritis, rheumatoid arthritis, gouty arthritis,psoriatic arthritis, traumatic rupture or detachment, achondroplasia,costochondritis, Spondyloepimetaphyseal dysplasia, spinal discherniation, lumbar disk degeneration disease, degenerative jointdisease, relapsing polychondritis, osteochondritis dissecans andaggrecanopathies. 48.-50. (canceled)
 51. A polypeptide cross-blockingbinding to ADAMTS5 by a polypeptide represented by any one of SEQ IDNO:s 2, 116, 19, 1, 3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13,14, 15 and 18, and/or is cross-blocked from binding to ADAMTS5 by atleast a polypeptide represented by any one of SEQ ID NO:s 2, 116, 19, 1,3, 6, 16, 17, 10, 11, 9, 5, 4, 7, 117, 8, 12, 13, 14, 15 and 18, whereinsaid polypeptide comprises at least one VH, VL, dAb, immunoglobulinsingle variable domain (ISVD) specifically binding to ADAMTS5, whereinbinding to ADAMTS5 modulates an activity of ADAMTS5.